Poly(alkylene oxide) amino acid copolymers and drug carriers and charged copolymers based thereon

ABSTRACT

Copolymers of poly(alkylene oxides) and amino acids or peptide sequences are disclosed, which amino acids or peptide sequences have pendant functional groups that are capable of being conjugated with pharmaceutically active compounds for drug delivery systems and cross-linked to form polymer matrices functional as hydrogel membranes. The copolymers can also be formed into conductive materials. Methods are also disclosed for preparing the polymers and forming the drug conjugates, hydrogel membranes and conductive materials.

This is a division of application Ser. No. 07/726,301, filed Jul. 5,1991, now U.S. Pat. No. 5,219,566, which is a continuation in-part ofapplication Ser. No. 07/549,494, filed Jul. 6, 1990, abandoned, thedisclosure of which is hereby incorporated herein by reference thereto.

BACKGROUND OF THE INVENTION

The present invention relates to copolymers of poly(alkylene oxides) andamino acids or peptide sequences, and more particularly to copolymers ofpolyalkylene oxides such as polyethylene glycol (PEG), with amino acidsor peptide sequences. The present invention also relates to conjugatesof such polymers formed with pharmaceutically active compoundscovalently bonded to the amino acid or peptide sequence of thecopolymer. The present invention further relates to ionically conductivematerials, hydrogel membranes and semi-interpenetrating polymer networksprepared from the copolymers of the present invention.

The conjugation of biologically active polypeptides with water-solublepolymers such as PEG is well-known. The coupling of biologically activeand pharmaceutically active peptides and polypeptides to PEG and similarwater-soluble polymers is disclosed by U.S. Pat. No. 4,179,377 to Daviset al. Polypeptides modified with PEG are disclosed as exhibitingdramatically reduced immunogenicity and antigenicity. The PEG conjugatesalso exhibit a wide range of solubilities and low toxicity, and havebeen shown to remain in the bloodstream considerably longer than thecorresponding native Compounds yet are readily excreted. The PEGconjugates have also been shown not to interfere with enzymatic activityin the bloodstream or the conformation of the polypeptides conjugatedthereto. Accordingly, a number of PEG-conjugates of therapeutic proteinshave been developed exhibiting reduced immunogenicity and antigenicityand longer clearance times, while retaining a substantial portion of theprotein's physiological activity.

Attention has also focused upon the conjugation of PEG with therapeuticdrugs. Gnanov et als., Macromolecules, 17, 945-52 (1984) observed thatthe attachment of PEG to various drugs led to prolonged pharmacologicalactivity.

As disclosed in the above-cited U.S. Pat. No. 4,179,337, the conjugationof PEG begins with functionalization of the terminal hydroxyl groups ofthe polymer prior to coupling with a ligand of biological relevance,although some ligands are capable of covalently bonding to the terminalhydroxyl groups without functionalization. The foregoing is alsodisclosed in Zalipsky et al., J. Macromol. Sci-Chem., A21, 839-845(1984); and Zalipsky et al., Eur. Polym. J., 19, 1177-1183 (1983). Oneof the limitations of PEG is that it has only two reactive end groupsavailable for functionalization. This is a particularly severe designlimitation for PEG chains of high molecular weight which contain only avery small number of reactive groups for any given weight of polymer. Tocircumvent this problem, several reaction schemes have been disclosed inwhich PEG chains were copolymerized with a variety of difunctionalco-monomers. For example, Graham et al., Makromol. Chem. Macromol.Symp., 19, 255-73 (1988) and Imai et als., Makromol. Chem. Rapid.Commun., 5, 47-51 (1984) disclose copolymers of poly(oxyethylene)dicarboxylic acids with aliphatic and aromatic amines. Block copolymersof PEG with polyesters are disclosed by Wang et als., J. MacromolSci-Chem., A26(2&3), 505-18 (1989). Block copolymers of PEG withpoly(L-proline) are disclosed by Jeon et als., J. Polym. Sci. Part APolym. Chem., 27, 1721-30 (1989). Block copolymers of PEG Withpoly(gamma-benzyl L-glutamate) are disclosed by Cho et al., Makromol.Chem., 191, 981-91 (1990). In these references, the use of the PEG blockcopolymers as biomaterials is suggested. Polyethylene glycols,cross-linked by copolymerization with triols and diisocyanates for usein the preparation of hydrogels and hydrogel membranes are disclosed byKimura et als., Macromolecules, 16, 1024-6 (1983), Ouchi et als., J.Macromol. Sci-Chem., A24(9), 1011-32 (1987), and Bos et al., Acta PharmTechnol., 33(3), 120-5 (1987). The hydrogel and hydrogel membranes havebeen investigated as potential materials for controlled drug delivery.However, none of the above-disclosed PEG copolymers have the desirablestructural feature of having multiple functional groups at regular,predetermined intervals that can be utilized for drug attachment orcross-linking reactions.

The preparation of PEG ionomers with phosphate diester linkages isdisclosed by Pretula et als., Macromol. Chem. Rapid Commun., 9, 731-7(1988), the apparently only known example of a strictly alternatingcopolymer of PEG. However, the reaction schemes developed by Pretularequire highly reactive intermediates that need to be handled withextreme care. Consequently, the resulting copolymers have apparently notyet found any practical applications.

PEG copolymers having multiple pendant functional groups at regularpredetermined intervals that can be utilized for drug attachment orcross-linking reactions would be highly desirable.

SUMMARY OF THE INVENTION

These needs are met by the present invention, which provides copolymersof poly(alkylene oxides) and amino acids or peptide sequences, whichamino acids or peptide sequences provide pendant functional groups atregular intervals within the polymer for drug attachment orcross-linking reactions. The resulting polymer is dominated by thedesirable properties of PEG, while the amino acid or peptide sequencesprovide biocompatible moieties having pendant functional groups for drugattachment or cross-linking.

Therefore, in accordance with one aspect of the present invention,polymers are provided that are copolymers of a poly(alkylene oxide) andan amino acid or peptide sequence. In a first embodiment of this aspectof the invention, a polymer is provided in which the poly(alkyleneoxide) and amino acid or peptide sequence are copolymerized by way ofhydrolytically stable urethane linkages. The polymer contains one ormore recurring structural units independently represented by Formula I:##STR1##

In Formula I, R₁ is a poly(alkylene oxide), and R₂ is an amino acid orpeptide sequence containing two amino groups and at least one pendantcarboxylic acid group. The pendant carboxylic acid group is not involvedin the polymerization process and is thus retained as a pendant group onthe polymer. This pendant functional group can be further derivatized(e.g., converted to a different functional group), used for crosslinkingor for the attachment of ligands, e.g., drugs. Preferably, R₂ isrepresented by Formula II: ##STR2## R₃ and R₄ are independently selectedfrom saturated and unsaturated, straight-chained and branched alkylgroups containing up to 6 carbon atoms and alkyl phenyl groups, thealkyl portions of which are covalently bonded to an amine and contain upto 6 carbon atoms. The values for a and b are independently zero or one.R₅ is independently selected from --NH-- or --NH--AA--, wherein --AA--is an amino acid or peptide sequence, with the proviso that --AA-- has afree N-terminus. D is a pendant functional group having a structurerepresented by ##STR3## When D is ##STR4## Y is selected from --OH,--NH--NH2, --O--R₆ --NH₂, --O--R6--OH, --NH--R6--NH₂, --NH--R₆ --OH,##STR5## a C-terminus protecting group and a derivative of apharmaceutically active compound covalently bonded to the pendantfunctional group by means of an amide bond in the case when in theunderivatized pharmaceutically active compound a primary or secondaryamine is present at the position of the amide bond in the derivatives;or an ester bond in the case when in the underivatized pharmaceuticallyactive compound a primary hydroxyl is present at the position of theester bond in the derivative. When D is ##STR6## Y is a derivative of apharmaceutically active compound covalently bonded to the pendantfunctional group by means of X, wherein X is a linkage selected from--NH--NH-- in the case when in the underivatized pharmaceutically activecompound an aldehyde or ketone is present at the position linked to thependant functional group by means of X; --NH--NH--, --NH--R₆ --NH--,--O--R₆ --NH--, --O--R₆ --O-- or --NH--R₆ --O-- in the case when in theunderivatized pharmaceutically active compound a carboxylic acid ispresent at the position linked to the pendant functional group by meansof X; and ##STR7## in the case when in the underivatizedpharmaceutically active compound a primary or second amine or primaryhydroxyl is present in the position linked to the pendant functionalgroup by means of X. R₆ is selected from alkyl groups containing fromtwo to six carbon atoms, aromatic groups, alpha-, beta-, gamma- andomega amino acids, and peptide sequences.

In a second embodiment of this aspect of the invention, a polymer isprovided in which a terminal amino groups and an amino acid or peptidesequence are copolymerized by way of hydrolytically stable amidelinkages in the case of the poly(alkylene oxide) having terminal aminogroups, and by way of hydrolyzable ester linkages in the case ofpoly(alkylene oxides) having terminal hydroxyl groups.

The polymer contains one or more recurring structural unitsindependently represented by Formula III:

    --L--R.sub.1 --L--R.sub.2 --                               (III)

R₁ is a poly(alkylene oxide), L is --O-- or --NH-- and R₂ is an aminoacid or peptide sequence containing two carboxylic acid groups and atleast one pendant amino group. As with the pendant group of Formula I,the pendant amino group is not involved in the polymerization processand is thus retained as a pendant group on the polymer that can befurther derivatized, used for crosslinking, or for the attachment ofligands. Preferably R₂ is represented by Formula IV: ##STR8##

R₃, R₄, a and b are the same as described above with respect to FormulaII. R₅ is independently ##STR9## wherein --AA-- is an amino acid orpeptide sequence, with the proviso that --AA-- has a free C-terminus.

D is a pendant functional group representing either --NHZ or --NH--X₁--Z. When D is --NHZ, Z is hydrogen, ##STR10## an N-terminus protectinggroup or a derivative of a pharmaceutically active compound covalentlybonded to the pendant functional group by means of an amide bond in thecase when in the underivatized pharmaceutically active compound acarboxylic acid group is present at the position of the amide bond inthe derivative.

When D is --NH--X₁ --Z, Z is a pharmaceutically active compoundcovalently bonded to the pendant function group by means of X₁. X₁ is alinkage selected from ##STR11## in the case when in the underivatizedpharmaceutically active compound a carboxylic acid is present at theposition linked to the pendant functional group by means of X₁ ; and##STR12## in the case when in the underivatized pharmaceutically activecompound a primary or secondary amine or primary hydroxyl is present atthe position linked to the pendant functional group by X₁. R₆ is thesame as described above with respect to the linkages of Formula I andII.

In a third embodiment of this aspect of the invention, a polymer isprovided in which a poly(alkylene oxide) having terminal amino groupsand an amino acid or peptide sequence having at least one hydroxyl groupare copolymerized by way of hydrolytically stable urethane linkages. Thepolymer contains one or more recurring structural units independentlyrepresented by Formula III, in which L is --NH-- and R₂ is an amino acidor peptide sequence having at least one activated hydroxyl group, onecarboxylic acid group when only one activated hydroxyl group is present,and at least one pendant amino group that can be further derivatized,used for crosslinking or for the attachment of ligands, like the pendantamino group of Formula IV. R₂ is preferably represented by Formula V:##STR13##

R₃, R₄, a, b and D are the same as described above with respect toFormula IV. R₅ is selected from: ##STR14## wherein --AA-- is the same asdescribed above with respect to Formula IV.

Unlike the first two embodiments of this aspect of the invention, thethird embodiment does not require the amino acid or peptide sequence tohave either two free amino groups or two free carboxylic acid groups.This makes available for use with the present invention natural aminoacids such as hydroxylysine, serine, threonine, thyroxine and tyrosine,which can be polymerized through their hydroxyl and carboxylic acidgroups, with the amino group remaining free as a pendant functionalgroup.

In accordance with another aspect of the present invention,polymerization processes are provided for the preparation of thecopolymers of the present invention. In a first embodiment of thisaspect of the present invention, an interfacial polymerization processis provided for the preparation of the polymers of Formula I in whichthe poly(alkylene oxide) and amino acid or peptide sequence arecopolymerized by means of stable urethane linkages. The process includesthe steps of intimately admixing a solution of an activatedpoly(alkylene oxide) in a water-immiscible organic solvent with an aminoacid or peptide sequence in an aqueous solution having a pH of at least8.0, which amino acid or peptide sequence has protected C-terminals andat least two free amino groups; and recovering from the organic solventthe resulting copolymer of the poly(alkylene oxide) and the amino acidor peptide sequence.

In accordance with a second embodiment of this aspect of the invention,a solution polymerization process is provided for the preparation of thepolymers of Formula III in which the poly(alkylene oxide) and amino acidor peptide sequence are copolymerized by way of hydrolytically stableamide or hydrolyzable ester linkages. The process includes the steps ofcontacting a hydroxyl-terminated or amino-terminated poly(alkyleneoxide) with an amino acid or a peptide sequence in an organic solvent inthe presence of coupling reagent and an acylation catalyst, which aminoacid or peptide sequence has at least two free carboxylic acid groups,with the proviso that when the poly(alkylene oxide) ishydroxyl-terminated, the amino acid or peptide sequence has protectedN-terminals. The resulting copolymer of the poly(alkylene oxide) withthe amino acid or peptide sequence is then recovered.

In accordance with a third embodiment of this aspect of the invention, asolution polymerization process is provided for the preparation ofpolymers according to Formula III in which L is --NH--. A poly(alkyleneoxide) having terminal amino groups is copolymerized with an amino acidor peptide sequence by way of urethane linkages formed with activatedhydroxyl groups.

The process includes the step of providing an amino acid or peptidesequence having at least one hydroxyl group and protected C-terminalsand activating the hydroxyl group in an organic solvent with anactivating reagent in the presence of an acylation catalyst. Theactivated hydroxyl groups are then reacted with an amino-terminatedpoly(alkylene oxide) in the organic solvent and the resulting copolymerof the poly(alkylene oxide) with the amino acid or peptide sequence isthen recovered. If the amino acid of peptide sequence has one hydroxylgroup, the copolymer will be polymerized by way of alternating urethaneand amide linkages. If the amino acid or peptide sequence has more thanone hydroxyl group available for activation, polymerization can beperformed exclusively through these groups by way of urethane linkagesand the carboxylic acid groups of the amino acid or peptide sequence canalso be protected and remain free as pendant functional groups.

In accordance with yet another aspect of the present invention, methodsare provided for preparing polymer conjugates of the copolymers of thepresent invention and pharmaceutically active compounds. Hydrolyticallystable conjugates are utilized when the pharmaceutical compound isactive in conjugated form. Hydrolyzable conjugates are utilized when thepharmaceutical compound is inactive in conjugated form. The propertiesof the poly(alkylene oxide) dominate the copolymer and conjugatethereof.

The pharmaceutically active compound can be directly conjugated to thependant functional group of the copolymer, or it may be conjugated bymeans of a bifunctional linker. The linker should contain a functionalgroup capable of coralently bonding with the pendant functional group ora functionalized derivative thereof, and a functional group capable ofcovalently bonding with the pharmaceutically active compound or afunctionalized derivative thereof. The linker should also contain aspacer moiety such as an aliphatic or aromatic moiety, amino acid orpeptide sequence. Examples of linkers include alkanol amines, diamines,hydrazines, and the like.

As will be readily appreciated by those of ordinary skill in the art,numerous combinations of functional groups on aliphatic, aromatic, aminoacid and peptide compounds exist that are capable of covalently bondingwith the pendant functional groups, pharmaceutically actice compoundsand functionalized derivatives thereof. However, once having the benefitof the disclosure contained in the within specification, those orordinary skill in the art will comprehend the types of compoundssuitable as being conjugate linkers.

When a linker compound is employed the order of reaction is notimportant. The linker may first be attached to the pendant functionalgroup of the copolymer and then attached to the pharmaceutically activecompound. Likewise, the linker may first be attached to thepharmaceutically active compound and then attached to the copolymer.

In a first embodiment of this aspect of the invention, a method isprovided for preparing a polymer conjugate of a pharmaceutically activecompound which compound prior to conjugation has an amino or hydroxylgroup, and a copolymer of a poly(alkylene oxide) and an amino acid orpeptide sequence, which amino acid or peptide sequence has, prior toconjugation, a pendant carboxylic acid group, by directly attaching thepharmaceutically active compound to the pendant functional groups of thecopolymer. The method includes the steps of contacting, in an organicsolvent, in the presence of an coupling reagent and an acylationcatalyst, the pharmaceutically active compound and the copolymer. Theresulting conjugate of the copolymer and the pharmaceutically activecompound is then recovered. A hydrolytically stable amide bond is formedwhen the pharmaceutically active compound has an amino group prior toconjugation, linking the pharmaceutically active compound to thecopolymer. When the pharmaceutically active compound has a hydroxylgroup prior to conjugation, a hydrolytically unstable ester bond isformed linking the pharmaceutically active compound to the copolymer.When the pharmaceutically active compound prior to conjugation has anamino group, the copolymer can optionally have activated pendantcarboxylic acid groups.

In a second embodiment of this aspect of the invention, a method isprovided for preparing a polymer conjugate of a pharmaceutically activecompound, which compound has a carboxylic acid group prior toconjugation, and a copolymer of a poly(alkylene oxide) and an amino acidor peptide sequence, which amino acid or peptide sequence has, prior toconjugation, a pendant carboxylic acid group or active ester thereof,using an alkanol amine linker. The method includes the steps ofreacting, in an aqueous solution, in the presence of a water-solublecoupling reagent, the pendant carboxylic acid group of the copolymerwith a alkanol amine, so that an alkanol amide of the carboxylic acidgroup is formed. The pharmaceutically active compound and the copolymerare then contacted in a suitable solvent so that an ester linkage isformed between the alkanol amide of the copolymer and the carboxylicacid group of the pharmaceutically active compound, and the resultingconjugate of the copolymer and the pharmaceutically active compound isthen recovered.

In accordance with the second embodiment, the order of reaction may bereversed, so that the alkanol amine is first reacted with the carboxylicacid group of the pharmaceutically active compound to form an alkanolamide of the carboxylic acid group. The pharmaceutically active compoundand the copolymer are then contacted in the organic solvent so that anester linkage is formed between the alkanol amide of thepharmaceutically active compound and the pendant carboxylic acid groupof the copolymer.

In a third embodiment of this aspect of the invention, a method isprovided for preparing a polymer conjugate of a pharmaceutically activecompound, which compound has a carboxylic acid group prior toconjugation, and a copolymer of a poly(alkylene oxide) and an amino acidor peptide sequence, which amino acid or peptide sequence has, prior toconjugation, a pendant carboxylic acid group or an active ester thereof,using a diamine linker. The method includes the steps of reacting, in anorganic solvent, in the presence of an activating reagent and anacylation catalyst, the copolymer and a diamine, so that an amino amideof the pendant functional group is formed, and then contacting, in theorganic solvent, the copolymer with the pharmaceutically activecompound. The resulting conjugate of the copolymer and thepharmaceutically compound is then recovered. As with the secondembodiment, the order of reaction may be reversed so that an amino amideis first formed with the carboxylic acid group of the pharmaceuticallyactive compound, which amino amide is then reacted with the pendantcarboxylic acid group of the copolymer.

Regardless of whether the amino amide is formed of the polymer pendantfunctional group or the pharmaceutically active compound, thepharmaceutically active compound may be reacted with an excess ofcopolymer, together with an additional quantity of the diamine, therebyconjugating the pharmaceutically active compound with the pendantcarboxylic acid groups by way of amido amide linkages and formingavailable amino amide linkages with unconjugated pendant carboxylic acidgroups. The available amino amide linkages of the copolymer conjugateare then further reacted, in the organic solvent, in the presence ofsodium borohydride or sodium cyanoborohydride, with a monoclonalantibody having oxidized carbohydrate moieties, so that the carbohydratemoieties covalently attach to the available amino amide linkages. Theresulting conjugate of the copolymer, the pharmaceutically activecompound and the monoclonal antibody is then recovered.

In a fourth embodiment of this aspect of the invention, a method isprovided for preparing a polymer conjugate of a pharmaceutically activecompound, which compound has an aldehyde, ketone or carboxylic acidgroup prior to conjugation, and a copolymer of a poly(alkylene oxide)and an amino acid or peptide sequence, which amino acid or peptidesequence, prior to conjugation, has a pendant carboxylic acid group,using a hydrazine linker. The method includes the steps of reacting, inan organic solvent, in the presence of a coupling reagent and anacylation catalyst, the copolymer with an alkyl carbazate, so that analkyl carbazate of the pendant functional group is formed, and thenconverting the alkyl carbazate to an acyl hydrazine. Thepharmaceutically active compound is then contacted in the organicsolvent with the copolymer, and the resulting conjugate of the copolymerand the pharmaceutically active compound is then recovered.

In accordance with this embodiment of the invention, thepharmaceutically active compound may be reacted with an excess ofcopolymer, so that fee acyl hydrazine groups remain as pendantfunctional groups. The method can then further include the step ofreacting, in the organic solvent, in the presence of sodium borohydride,the pendant acyl hydrazine groups with a monoclonal antibody havingoxidized carbohydrate moieties, so that the oxidized carbohydratemoieties form diacyl hydrazides with the pendant functional group. Theresulting conjugate of the copolymer, the pharmaceutically activecompound and the monoclonal antibody is then recovered.

In a fifth embodiment of this aspect of the invention, a method isprovided for preparing a polymer conjugate of a pharmaceutically activecompound, which compound has a carboxylic acid group prior toconjugation, and a copolymer of a poly(alkylene oxide) and an amino acidor peptide sequence, which amino acid or peptide sequence has a pendantamino group prior to conjugation, by directly attaching thepharmaceutically active compound to the pendant functional group of thecopolymer. The method includes the steps of reacting, in an organicsolvent, in the presence of an activating reagent and an acylationcatalyst, the pharmaceutically active compound and the copolymer, andthen recovering the resulting conjugate of the copolymer and thepharmaceutically active compound. In accordance with this embodiment ofthe invention, the pharmaceutically active compound may be reacted withan excess of the copolymer, so that pendant amino groups remain. Themethod then further includes the step of reacting, in the organicsolvent, in the presence of sodium borohydride, the remaining pendantamino groups with a monoclonal antibody having oxidized carbohydratemoieties, so that the oxidized carbohydrate moieties covalently attachto the pendant amino groups. The resulting conjugate of the copolymerwith the pharmaceutically active compound and the monoclonal antibody isthen recovered.

In accordance with still yet another aspect of the present invention, aconductive composition is provided of an alkali metal electrolyte saltcombined with a copolymer of a poly(alkylene oxide) and an amino acid orpeptide sequence, which amino acid or peptide sequence has pendantcarboxylic acid groups protected by C-terminus protecting groups.Preferably, the alkali metal electrolyte salt is a lithium salt selectedfrom LiAsF₆, LiPF₆, LiI, LiBr, LiBF₆, LiAlCl₄, LiCF₃ CO₂ and LiCF₃ SO₃.

According to another aspect of the present invention, the conductivecomposition of the present invention is utilized as a solid electrolytein an electrochemical cell. The electrochemical cell includes a cathode,an anode and the conductive material of the present invention. Thecathode includes a cathode-active material capable of intercalatinglithium and the anode is preferably a counter-electrode capable ofintercalating lithium. More preferred embodiments utilize a lithiatedtransition metal chalcogenide as the cathode-active material and agraphitic carbon as the counter-electrode.

Still yet another aspect of the present invention provides hydrogelmembranes and semi-interpenetrating polymer networks prepared from thepolymers of the present invention. The hydrogel membranes have highequilibrium water content and good mechanical strength, and, as such,are suitable for many biomedical applications such as wound dressingsand implants.

One embodiment of this aspect of the present invention provides hydrogelmembranes of polymer matrices formed from copolymers of poly(alkyleneoxides) and amino acids or peptide sequences, cross linked by way ofurethane linkages between a trifunctional amine and the poly(alkyleneoxide) moiety of the copolymer. The urethane linkages are non-degradableunder physiological conditions. The cross link density of the membranecan be controlled by varying the length of the poly(alkylene oxide)chain used in the cross linking reaction.

A second embodiment of this aspect of the present invention provideshydrogel membranes of polymer matrices formed from copolymers ofpoly(alkylene oxides) and amino acids or peptide sequences, which aminoacids or peptide sequences have pendant acyl hydrazine groups. Thecopolymers are cross linked by way of hydrolytically labile acylsemicarbazide linkages between a diisocyanate and the pendant acylhydrazine groups of the polymer. Hydrogel membranes of this aspect ofthe present invention when incorporated with water, demonstrate highwater content and high mechanical strength.

A third embodiment of this aspect of the present invention providessemi-interpenetrating polymer networks (IPN) of a linear, preformedsecond polymer entrapped within the polymer matrices of the presentinvention. The second polymer is chosen to be biocompatible and toimprove a physical characteristic, such as tensile strength, of thepolymer matrix. Polymers that are ordinarily immiscible may be combinedto form the semi-IPN's of the present invention. The semi-IPN's of thepresent invention can be formed from polymers that would not bephysically blendable by any other means. According to preferred aspectsof this embodiment of the invention, the second polymer is poly(BPAcarbonate) or poly(desaminotyrosyl tyrosine hexyl ester carbonate).

Still yet another aspect of the present invention provides methods bywhich the hydrogel membranes and semi-IPN's of the present invention maybe prepared. According to one embodiment of this aspect of the presentinvention, a method is provided for preparing a cross linked polymermatrix of a copolymer of a poly(alkylene oxide) and an amino acid orpeptide sequence, wherein at least one terminus of the copolymer is thepoly(alkylene oxide). The method includes the steps of providing a firstsolution of the copolymer dissolved in an organic solvent in which thepolymer matrix is soluble, protecting the pendant C-terminals orN-terminals of the amino acid or peptide sequence of the copolymer andthen forming in the first solution an active ester of the poly(alkyleneoxide) terminus of the copolymer. The first solution is then mixed witha second solution of an equivalent quantity of a trifunctional amine ina solvent in which the polymer matrix is soluble so that urethanelinkages form between the active ester and the tris(amino) amine. Theresulting cross-linked copolymer polymer matrix is then recovered. Inaccordance with this embodiment of this aspect of the present invention,a method is also provided for preparing a semi-IPN by .first dissolvinga linear, pre-formed second polymer in the first solution before mixingthe first solution with the second solution so that the second polymeris entrapped within the cross-linked polymer matrix, as the polymermatrix is formed.

According to a second embodiment of this aspect of the presentinvention, a method is provided for preparing a cross-linked polymermatrix of a copolymer of a poly(alkylene oxide) and an amino acid orpeptide sequence, which amino acid or peptide sequence has a pendantacyl hydrazine group. The method includes the steps of providing asolution of the copolymer in an organic solvent in which the polymermatrix is soluble and adding an equivalent quantity of diisocyanate tothe solution so that acyl semicarbazide linkages form between thependant acyl hydrazines and the diisocyanate. The resulting cross-linkedcopolymer polymer matrix is then recovered. In accordance with thisembodiment of this aspect of the present invention, a method is alsoprovided for preparing a semi-IPN by dissolving a linear, pre-formedsecond polymer in the first solution before mixing the first solutionwith the second solution so that the second polymer is trapped withinthe cross-linked copolymer matrix as the polymer matrix is formed.

It can be readily appreciated that the present invention provides aversatile family of poly(alkylene oxide) copolymers having multiplependant functional groups at regular predetermined intervals. By beingcapable of forming linkages through the pendant functional groups, whichlinkages have varying degrees of hydrolytic stability or instability,the copolymers are useful for a variety of biomedical end-useapplications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the weight loss with time of a hydrogel membrane of thepresent invention in phosphate buffer (pH 7.4) at 60° C.

FIG. 2 depicts an Arrhenius plot of conductivity versus temperature foran ionically conductive material of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The polymers of the present invention are copolymers of poly(alkyleneoxides) and amino acids or peptide sequences. The polymers thus includeone or more recurring structural units in which the poly(alkylene oxide)and the amino acid or peptide sequence are copolymerized by means ofurethane linkages, which structural units are independently representedby Formula I disclosed above. With respect to Formula I, R₁ is apoly(alkylene oxide) and R₂ is an amino acid or peptide sequencecontaining two amino groups and at least one pendant carboxylic acidgroup.

The poly(alkylene oxides) suitable for use in the polymers of thepresent invention include polyethylene glycol (PEG), polypropyleneglycol, poly(isopropylene glycol), polybutylene glycol, poly(isobutyleneglycol) and copolymers thereof. Preferred poly(alkylene oxides) for usewith the present invention have the structure:

    --(O--R.sub.8).sub.c --(O--R.sub.9).sub.d --(O--R.sub.10).sub.e --

wherein R₈, R₉ and R₁₀ are independently selected from straight-chainedand branched alkyl groups containing up to 4 carbon atoms, c is aninteger between about 1 and about 100, inclusive, and d and e areindependently integers between 0 and about 100, inclusive, with theproviso that the sum of c, d and e is between about 10 and about 100,inclusive.

The most preferred poly(alkylene oxide) is PEG.

The molecular weight of the poly(alkylene oxide) is not critical, andwould depend mainly upon the end use of a particular copolymer. Those ofordinary skill in the art are capable of determining molecular weightranges suitable for their end-use applications. In general, the usefulrange of molecular weight is a number average molecular weight betweenabout 600 and about 200,000 daltons, and preferably between about 2,000and about 50,000 daltons. Because the copolymers are hydrolyticallystable, lower molecular weight polyalkylene oxides are preferred toinsure that the resulting polymer is not too large to be eliminated bythe kidney. Preferably, the molecular weight of the resulting polymershould not exceed 50,000 daltons.

The amino acid or peptide sequence represented by R₂ in Formula Ipreferably has a structure according to Formula II wherein R₃ and R₄ areindependently selected from saturated and unsaturated, straight-chainedand branched alkyl groups containing up to 6 carbon atoms andalkylphenyl groups, the alkyl portions of which are covalently bonded toan amine and contain up to 6 carbon atoms. Included within thedefinition of the alkyl or phenyl portions of the alkyl phenyl groupsare alkyl or phenyl groups substituted by one or more substituentsselected from hydroxyl, halogens, amino, and the like. The values for aand b are independently 0 or 1. R₅ is --NH-- or --NH--AA--, wherein--AA-- is an amino acid or peptide sequence, with the proviso that--AA-- contains a free N-terminus so that, when present, R₂ represents apeptide sequence of two or more amino acids.

The polymers of Formula I possess pendant functional groups at regularintervals within the polymer having the structure: ##STR15##

When D is ##STR16## and Y is --OH, the pendant functional groups arecarboxylic acid groups. The pendant carboxylic acid groups may befurther functionalized, in which case Y is selected from --NH--NH2,--OR₆ --NH2, --OR₆ --OH, --NH--R₆ --NH₂, --NH--R₆ --OH, and: ##STR17##

Y can also be a C-terminus protecting group or a derivative of apharmaceutically active compound covalently bonded to the recurringstructural unit by the pendant functional group. R₆ is selected fromalkyl groups containing from 2 to 6 carbon atoms, aromatic groupsalpha-, beta-, gamma- and omega amino acids, and peptide sequences.

With respect to the amino acid or peptide sequence represented by R₂ inFormula I and having a structure according to Formula II, R₃ and R₄ arepreferably alkyl groups containing from 1 to 4 carbon atoms, inclusive.When R₂ is an amino acid, R₅ is --NH--. When R₂ is a peptide sequence,R₅ is --NH--AA--, wherein the --AA-- of R₅ is bonded to R₃ or R₄ by wayof the --NH-group of R₅. The single amino acids and the two or moreamino acids making up the peptide sequences are preferably alpha aminoacids, in which case either a or b, or both, is zero, and --AA--represents one or more alpha amino acids. Even more preferably, theamino acids and the two or more amino acids making up the peptidesequences are natural amino acids, in which instance, R₃ (when b iszero) or R₄ (when a is zero) is --CH₂ --CH₂ --CH₂ -- in the case ofornitine --CH₂ --CH₂ --CH₂ --CH₂ --in the case of lysine, ##STR18## inthe case of cystine, and ##STR19## in the case of hydroxylysine, and--AA-- represents one or more natural amino acids.

The peptide sequences of R₂ are preferably sequences containing from 2to about 10 amino acid residues, in which case --AA-- would preferablycontain from 1 to about 9 amino acid residues. The peptide sequences ofR₂ even more preferably contain from 3 to 7 amino acid residues,inclusive, in which case, --AAwould contain from 2 to 6 amino acidresidues, inclusive.

As noted above, Y of the pendant functional group can be a C-terminusprotecting group. C-terminus protecting groups are well-known to thoseof ordinary skill in the art and include those disclosed in Bodanszky,The Practice of Peptide Synthesis (Springer-Verlag, New York, 1984), thedisclosure of which is herein incorporated by reference thereto.Preferred C-terminus protecting groups are alkyl, aryl and siliconprotecting groups.

As noted above, the pendant carboxylic acid groups may be furtherfunctionalized. In such a case, Y is preferably --NH--NH₂. However, R₆,when present, is preferably an ethyl group, a natural alpha-amino acidor a peptide sequence containing from 2 to 10 natural amino acidresidues.

As also disclosed above, Y can be a derivative of a pharmaceuticallyactive compound covalently bonded to the recurring structural unit bymeans of the pendant functional group. Y is covalently bonded to therecurring structural unit by means of an amide bond in the case when inthe underivatized pharmaceutically active compound a primary orsecondary amine is present at the position of the amide bond in thederivative. Examples of underivatized pharmaceutically active compoundscontaining a primary or secondary amine include acyclovir, cephradine,melphalan, procaine, ephedrine, adriamycin, daunomycin, and the like.

Y is covalently bonded to the recurring structural unit by means of anester bond in the case when in the underivatized pharmaceutically activecompound a primary hydroxyl is present at the position of the ester bondin the derivative. Examples of underivatized pharmaceutically activecompounds containing a primary hydroxyl group include acyclovir,plumbagin, atropine, quinine, digoxin, quinidine and the like, as wellas biologically active peptides.

Y can also be a derivative of a pharmaceutically active compoundcovalently bonded to the recurring structural unit by means of --X--, sothat the pendant functional group has the structure: ##STR20## X is alinkage derived from the above-described further functionalized pendantcarboxylic acid groups. X is --NH--NH-- in the case when in theunderivatized pharmaceutically active compound an aldehyde or ketone ispresent at the position linked to the pendant functional group of therecurring structural unit by means of X. Examples of underivatizedpharmaceutically active compounds containing an aldehyde or ketoneinclude adriamycin, daunomycin, testosterone, and the like. Steroidssuch as ketones and aldehydes are also easily generated by conventionalmethods.

X is --NH--NH--, --NH--R₆ --NH--, --O--R₆ --NH--, --O--R₆ --O-- or--NH--R₆ --O-- in the case when in the underivatized pharmaceuticallyactive compound a carboxylic acid is present at the position linked tothe pendant functional group of the recurring structural unit by meansof X. Examples of underivatized pharmaceutically active compoundscontaining a carboxylic acid include chlorin e₆, cephradine,cephalothin, melphlan, penicillin V, aspirin, nicotinic acid,chemodeoxycholic acid, chlorambucil, and the like, as well asbiologically active peptides.

In the case when in the underivatized pharmaceutically active compound aprimary or secondary amine or primary hydroxyl is present in theposition linked to the pendant functional group of the recurringstructural unit by means of X, X is: ##STR21## Examples of suchunderivatized pharmaceutically active compounds include those compoundslisted above with respect to amide and ester linkages.

When X is --NH--NH--, --NH--R₆ --NH-- or --O--R₆ --NH--, Y can also be aderivative of a monoclonal antibody having oxidized carbohydratemoieties in the case when in the underivatized oxidized monoclonalantibody a ketone or aldehyde is present at the position linked to therecurring structural unit by means of X. In this embodiment, the polymerpreferably contains a recurring structural units having an oxidizedmonoclonal antibodies covalently bonded thereto at the pendantfunctional group and recurring structural units having a derivative of apharmaceutically active compound covalently bonded thereto at thependant functional group. The monoclonal antibody and thepharmaceutically active compound are preselected so that the monoclonalantibody targets cells for which it is specific for treatment by thepharmaceutically active compound it is co-conjugated with. For example,chlorin e₆ is a photosensitizer that can be co-conjugated with an anti-Tcell monoclonal antibody to target the photosensitizer to T-cellleukemia cells.

Only one monoclonal antibody is required to be bound to a polymer tobind the polymer to a cell for which the monoclonal antibody isspecific. The ratio of pharmaceutically active compound to monoclonalantibody should be between about 4 and about 100. Preferably, the ratiois between about 6 and about 20.

Alternatively, the polymers of the present invention can have one ormore recurring structural units in which the poly(alkylene oxide) andthe amino acid or peptide sequence are copolymerized by means of amideor ester linkages, which structural units are independently representedby Formula III disclosed above. With respect to Formula III, R₁ is apoly(alkylene oxide), L is --O-- or --NH-- and R₂ is an amino acid orpeptide sequence containing two carboxylic acid groups and at least onependant amino group. The poly(alkylene oxides) of R₁, and the preferredspecies of same, are the same as described above with respect to FormulaI. However, because the amide and ester linkages are hydroslyticallylabile, there is no preference for limiting the molecular weight of thepoly(alkylene oxide) below 50,000 daltons.

R₂ is preferably an amino acid or peptide sequence having a structureaccording to Formula IV disclosed above, wherein R₃, R₄, a and b are thesame as described above with respect to Formula II. R₅ is selected from:##STR22## wherein --AA-- is an amino acid or peptide sequences, with theproviso that --AA-- contains a free C-terminus, so that when present, R₂represents a peptide sequence of two or more amino acids.

The polymers of Formula III also possess pendant functional groups atregular intervals within the polymer, having the structure --NHZ or--NH--X₁ --Z. When D is --NHZ and Z is hydrogen, the pendant functionalgroups are amino groups. As with the pendant carboxylic acid groups ofthe polymers of Formula I, the pendant amino groups may be furtherfunctionalized, in which case Z is selected from: ##STR23## Z can alsobe an N-terminus protecting group or a derivative of a pharmaceuticallyactive compound covalently bonded to the recurring structural unit bythe pendant functional group. R₆ and the preferred species thereof arethe same as described above with respect to Formula II.

With respect to the amino acid or peptide sequence represented by R₂ inFormula III and having a structure according to Formula IV, R₃ and R₄are again preferably alkyl groups containing from 1 to 4 carbon atoms,inclusive. When R₂ is an amino acid, R₅ is a carboxyl group. When R₂ isa peptide sequence, R₅ is: ##STR24## wherein the --AA-- of R₅ is bondedto R₃ or R₄ by way of the carbonyl group of R₅.

The single amino acids and the two or more amino acids making up thepeptide sequences are preferably alpha-amino acids, in which case a orb, or both, is zero, and --AA-- represents one or more alpha-aminoacids. More preferably, the single amino acids and the two or more aminoacids making up the peptide sequences are natural amino acids, in whichinstance R₃ (when b is zero) or R₄ (when a is zero) is --CH₂ -- in thecase of aspartic acid, --CH₂ --CH₂ -- in the case of glutamic acid, and##STR25## in the case of cystine.

When present, --AA-- would then represent one or more natural aminoacids. For --AA--, the peptide sequence lengths and preferred valuestherefore are the same as described above with respect to Formulas I andII.

As noted above, Z of the pendant amino group of the recurring structuralunit can represent a N-terminus protecting group. N-terminus protectinggroups are well-known to those of ordinary skill in the art and includethose disclosed in the above-cited Bodanszky, The Practice of PeptideSynthesis, the disclosure of which is herein incorporated by referencethereto. The preferred N-terminus protecting groups arebenzyloxycarbonyl and tert-butoxycarbonyl groups.

As also noted above, Z could also be a derivative of a pharmaceuticallyactive compound covalently bonded to the recurring structural unit bythe pendant functional group. Z is covalently bonded to the recurringstructural unit by means of an amide bond in the case when in theunderivatized pharmaceutically active compound a carboxylic acid groupis present in the position of the amide bond in the derivative. Examplesof underivatized pharmaceutically active compounds containing carboxylicacid groups include those described above for Y with respect to FormulaII.

Z can also be a derivative of a pharmaceutically active compoundcovalently bonded to the recurring structural unit by means of --X₁ --,so that the pendant functional group has the structure --NH--X₁ --Z. X₁is a linkage derived from the above-described further functionalizedpendant amino groups. X₁ is a linkage selected from: ##STR26## in thecase when in the underivatized pharmaceutically active compound acarboxylic acid is present at the position linked to the pendantfunctional group of the recurring structural unit by means of X₁. Asnoted above, examples of underivatized pharmaceutically active compoundscontaining carboxylic acid groups have been previously listed.

In the case when in the underivatized pharmaceutically active compound aprimary or secondary amine or primary hydroxyl is present at theposition linked to the pendant functional group of the recurringstructural unit by X₁, X₁ is: ##STR27## of underivatizedpharmaceutically active compounds containing a primary or secondaryamine or primary hydroxyl are the same as those listed above for Y withrespect to Formula II.

Z can also be a derivative of a monoclonal antibody having oxidizedcarbohydrate moieties covalently bonded to the pendant amino group ofthe recurring structural unit by means of an amide bond in the case whenin the underivatized oxidized monoclonal antibody a ketone or aldehydeis present at the position of the amide bond in the derivative. As withthe polymer having carboxylic acid pendant functional groups, thepolymer having pendant amino groups preferably contains both recurringstructural units having oxidized monoclonal antibodies covalently bondedthereto at the pendant functional group and recurring structural unitshaving a derivative of a pharmaceutically active compound covalentlybonded thereto at the pendant functional group, with the monoclonalantibody and the pharmaceutically active compound preselected so thatthe monoclonal antibody targets cells for which it is specific fortreatment by the pharmaceutically active compound it is co-conjugatedwith.

As noted above, when L is --NH--, R₂ can also be an amino acid orpeptide sequence having at least one activated hydroxyl group, onecarboxylic acid group when only one activated hydroxyl group is present,and at least one pendant amino group. Preferably R₂ has the structure ofFormula V, in which R₃, R₄, a, b, Z and AA and the preferred speciesthereof are the same as disclosed above for Formula IV and R₅ isselected from: ##STR28## When R₂ is a natural amino acid, R₃ (when b iszero) or R₄ (when a is zero) is --CH₂ -- in the case of serine, and:##STR29## in the case of tyrosine.

The polymers of the present invention can also have both the amide andester recurring structural units of Formula III, so that, with respectto Formula III, L is --O-- for some recurring structural units and--NH-- for other recurring structural units. By varying the ratio of--O-- and --NH--, the hydrolytic stability of the polymer can betailored to suit the needs of the end-use application.

The polymers of Formulas I and III have an absolute weight averagemolecular weight in the range of from about 10,000 to about 200,000daltons, with about 20,000 to about 50,000 daltons being preferred fordrug conjugate end-use applications. Molecular weights are determined bygel permeation chromatography relative to polyethylene glycol. Statedanother way, the polymers of the present invention have from about 10 toabout 100 repeating units represented by one of the structures ofFormulas I and III, depending upon the molecular weight of thepoly(alkylene oxide) used. As noted above, the molecular weight of thepolymer should preferably not exceed 50,000 daltons, when the backboneof the polymer is not hydrolytically labile.

Interfacial Polymerization

The polymers of Formula I are prepared by an interfacial polymerizationprocess in which the poly(alkylene oxide) and amino acid or peptidesequence are copolymerized by means of stable urethane linkages. Theinterfacial polymerization utilizes a water-immiscible organic solutioncontaining one or more activated poly(alkylene oxides). Thepoly(alkylene oxides) are described above and include compoundsspecifically enumerated as preferred. Activated poly(alkylene oxides)and the preparation of same, are well-known to those of ordinary skillin the art. For example, poly(alkylene oxides) can be activated byreaction with cyanuric chloride, or by succinylation of terminalhydroxyl groups followed by dicyclohexylcarbodiimide-mediatedcondensation with N-hydroxy succinimide, or by the formation ofimidazolyl formate derivatives using carbonyl diimideazole, or byreaction with chloroformates of 4-nitrophenol and 2,4,5-trichlorphenol.

The preferred activated form of the poly(alkylene oxide) is thesuccinimidyl carbonate prepared by reacting the terminal hydroxyl groupsof the poly(alkylene oxide) with phosgene to form the chloroformate,which is then reacted with N-hydroxy succinimide to form thesuccinimidyl carbonate. The preparation of poly(alkylene oxide)succinimidyl carbonates is described in co-pending U.S. patentapplication Ser. No. 340,928 by Zalipsky, filed Apr. 19, 1989, nowabandoned, the disclosure of which is hereby incorporated herein byreference thereto.

The solution of the active carbonate of the poly(alkylene oxide) in theorganic solvent is added to an aqueous solution containing one or moreof the amino acids or peptide sequences described above, includingcompounds specifically enumerated as preferred, having protectedC-terminals and at least two free amino groups. The aqueous solution isbuffered to a pH of at least 8.0. Suitable buffers include NaHCO₃ andNa₂ CO₃. The organic solution is added to the aqueous solution withvigorous stirring, which stirring is continued for several hours betweenabout 4° C. and about 40° C. and preferably at ambient temperature.Slightly higher or lower temperatures are also suitable, depending uponthe requirements of the reactants, which can be readily determined bythose of ordinary skill in the art without undue experimentation. Theactivated poly(alkylene oxide) reacts with the amino acid or peptidesequence to produce the copolymer of Formula I. The mixture is thenacidified to a pH of about 2.0 or lower. The two phases separate, withthe organic phase containing the polymer.

The reaction rate is a function of the concentration of the two phases,with the reaction rate increasing as phase concentration increases.Therefore, while dilute phase concentrations are operative, higherconentrations are preferred to accelerate the reaction rate. The onlyupper limit to phase concentration is the solubility of the reactants ineach phase. Examples of suitable water-immiscible organic solventsinclude methylene chloride, chloroform, dichloroethane and the like.Equimolar ratios of activated poly(alkylene oxide) to amino acid orpeptide sequence starting materials are employed to maximize polymerlength.

After the phases separate, the organic phase is washed with 1N HClfollowed by washing with saturated NaCl . The organic layer is thendried over anhydrous MgSO₄, filtered and concentrated. The polymer isprecipitated using cold ether.

The polymer can then be purified by conventional purificationtechniques, Such as by dialysis against distilled water with a molecularweight sizing membrane or by elution with a molecular weight sizingchromatography column.

Solution Polymerization--First Mode

The polymers of Formula III are prepared by a solution polymerizationprocess in which the poly(alkylene oxide) and the amino acid or peptidesequence are copolymerized by means of hydrolytically stable amide orhydrolyzable ester linkages. The poly(alkylene oxide) should first bedried by the azeotropic removal of water by distillation in toluene,followed by drying under vacuo. The solution polymerization is carriedout in an organic solvent such as methylene chloride, chloroform,dichloroethane and the like.

The poly(alkylene oxides) utilized in the reaction can have eitherhydroxyl terminals or amino terminals and are otherwise as describedabove and include compounds specifically enumerated as preferred. Thepoly(alkylene oxide) is dissolved in the solvent and stirred underargon. An equimolar quantity is then added of one or more of the aminoacids or peptide sequences described above, including compoundsspecifically enumerated as preferred, having protected N-terminals. Thereaction mixture may be heated slightly to dissolve the amino acid orpeptide. The solution concentration of either compound is not critical.An excess quantity of a coupling reagent is also added to the reactionmixture, together with an excess quantity of an acylation catalyst.Suitable coupling reagents and the quantities to employ are well-knownand disclosed by the above-cited Bodanszky, Principles of PeptideSynthesis, the disclosure of which is hereby incorporated herein byreference thereto.

Examples of such coupling reagents include, but are not limited to,carbodiimides such as ethyl dimethylaminopropyl carbodiimide (EDC),diisopropyl carbodiimide and 3-[2-morpholinyl-(4)-ethyl]carbodiimide,p-toluene sulfonate, 5-substituted isoxazolium salts, such as Woodward'sReagent K, and the like. Suitable acylation catalysts and the quantitiesto employ are also well-known, and include, but are not limited to,dimethylaminopyridinium toluene sulfonate, hydroxybenzotriazole,imidazoles, triazole, dimethyl amino pyridene, and the like.

The reaction mixture is then stirred between about 4° C. and about 40°C. and preferably at room temperature until completion of the reaction,typically within 24 hours, usually overnight.

The poly(alkylene oxide) reacts with the amino acid or peptide sequenceto produce the copolymer of Formula III. A urea precipitate is removedby filtration, and the polymer is then precipitated with cold ether,filtered and dried under vacuum. The polymer can then be furtherpurified by conventional methods, typically by reprecipitation fromisopropanol.

Solution Polymerization--Second Mode

The polymers of Formula III can also be prepared by a solutionpolymerization process in which a poly(alkylene oxide) having aminoterminals and an amino acid or peptide sequence having at least onehydroxyl group are copolymerized in an organic solvent by means ofhydrolytically stable urethane linkages. The one or more hydroxyl groupsof the amino acid or peptide sequence should first be activated in theorganic solvent. The activation step is well-known and essentiallyconventional. For example, the hydroxyl group can be activated byreacting it with an alkyl chloroformate, or with p-nitrophenylchloroformate. Alternatively, the hydroxyl group can be activated asdescribed above with respect to the activation of poly(alkylene oxides)for the interfacial polymerization process of the present invention,preferably utilizing the process disclosed by U.S. patent applicationSer. No. 340,928, now abandoned by Zalipsky, incorporated herein byreference thereto. Amino and carboxylic acid groups that are not toparticipate in the copolymerization, but rather are to serve as pendantfunctional groups, should be protected.

The activation is carried out in the presence of one or more of theactivating reagents and acylation catalysts described above with respectto the first mode of solution polymerization. The same ratio ofactivating reagent and acylation catalyst to amino acid or peptidesequence should also be utilized. The same solvents are utilized asdescribed above with respect to the first mode solution polymerization.

The poly(alkylene oxide) should first be dried as described above withrespect to the first mode solution polymerization. After the activationof the one or more hydroxyl groups of the amino acid or peptide sequenceis complete, the poly(alkylene oxide) is then added to the reactionmixture with stirring. As with the first mode solution polymerizationequimolar quantities of reactants are preferred. The reaction mixture isthen stirred at room temperature until completion of the reaction,typically within 24 hours, usually overnight.

The poly(alkylene oxides) utilized in the reaction have amino terminalsand are otherwise as described above and include compounds specificallyenumerated as preferred. The poly(alkylene oxide) reacts with theactivated hydroxyl groups of the amino acid or peptide sequence to formurethane linkages. When the amino acid or peptide sequence contains onlyone hydroxyl group to be activated, a carboxylic acid group is also leftunprotected and the poly(alkylene oxide) reacts with this carboxylicacid group as in the first mode solution polymerization to form an amidelinkage.

The resulting polymer is then precipitated, separated and purified asdescribed above with respect to the first mode solution polymerization.

The polymers of the present invention can be used in the preparation ofdrug carriers by conjugating the pendant functional groups eitherdirectly with reactive functional groups on a drug molecule, or by firstfurther functionalizing the pendant functional group to improve itsreactivity with or selectivity for a functional group on a candidatedrug molecule. Accordingly, the copolymers of the present invention canbe conjugated with candidate drug molecules by one of the modes ofconjugation set forth below.

Drug Conjugation--First Mode

The polymers of Formula I, having pendant carboxylic acid groups, can bedirectly conjugated with pharmaceutically active compounds that, priorto conjugation, have an amino or hydroxyl group. The polymers of FormulaI are described above, and include polymers specifically enumerated aspreferred. Pharmaceutically active compounds having amino or hydroxylgroups are also described above.

The conjugation reaction utilizes an organic solvent in which thereactants are soluble. Examples of suitable organic solvents includeDMF, CH₃ CN, CH₂ Cl₂, and the like. The appropriate quantities of thepolymer and the pharmaceutically active compound are dissolved in thesolvent. The solvent may be heated slightly to dissolve the reactants.An excess of the pharmaceutically active compound is preferred to insuresubstantial conjugation of the pendant functional groups of thepolymers. The total solution concentration (w/v %) of both compoundscombined is not critical, and will vary depending upon the solubility ofthe materials. Complete solubility of the pharmaceutically activecompound is also not critical, because the compound will be solubilizedupon conjugation with the polymer. An activating reagent and anacylation catalyst are also added to facilitate the reaction asdescribed above with respect to the interfacial polymerization.

The reaction mixture is then stirred between about 4° C. and about 40°C. until completion of the reaction, typically within 24 hours, usuallyovernight. Temperatures between about 15° C. and about 25° C. are morepreferred and temperatures close to or below room temperature are evenmore preferred to preserve the integrity of the biologically activemolecule and to minimize side reactions.

A urea product precipitates, which is removed by filtration. The polymerconjugate is then precipitated with a solvent in which the polymer haspoor solubility, e.g., ether, hexane and the like, filtered and purifiedby further reprecipitation crystalization, from such solvent as ethanol,ethyl acetate, iso-propanol and the like. The product then is dried invacuo.

As noted above, when the pharmaceutically active compound has a hydroxylgroup prior to conjugation, a hydrolytically unstable ester bond isformed linking the pharmaceutically active compound to the copolymer bymeans of the pendant functional group. When the pharmaceutically activecompound has an amino group prior to conjugation, a hydrolyticallystable amide bond is formed linking the pharmaceutically active compoundto the copolymer by means of the pendant functional group. If thepharmaceutical compound is active in conjugated form, then ahydrolytically stable bond is desirable. However, if the pharmaceuticalcompound is inactive in conjugated form, then a hydrolytically unstablebond is desirable. When the pharmaceutically active compound has both anamino and a hydroxyl group, the question of which group to conjugate towill thus depend upon the activity of the pharmaceutical compound inconjugated form. Once a decision is made to conjugate to either theamino group or the hydroxyl group, the group through which conjugationis not to occur should be protected to prevent the formation ofundesirable conjugates. The attachment of such protective groups iswell-known to those of ordinary skill in the art.

When the pharmaceutically active compound prior to conjugation has anamino group, the pendant carboxylic acid group at the copolymer ispreferably an activated pendant carboxylic acid group. The activation ofsuch carboxylic acid groups is well-known and essentially conventional.For example, the pendant carboxylic acid group can be reacted withN-hydroxy succinimide in the presence of a coupling agent such asdicyclohexyl carbodiimide in a solvent such as DMF, CHCl₃, pyridine andthe like.

Drug Conjugation--Second Mode

The polymers of Formula I having pendant carboxylic acid groups can alsobe conjugated with pharmaceutically active compounds that, prior toconjugation, have a carboxylic acid group, by first reacting the pendantcarboxylic acid group of the copolymer with a alkanol amine, so that anamide of the pendant carboxylic acid group is formed. The polymers ofFormula I are disclosed above and include polymers specificallyenumerated as preferred. Pharmaceutically active compounds having acarboxylic acid group are also described above.

Pharmaceutically active compounds having a carboxylic acid group canalso be formed from pharmaceutically active compounds having hydroxylgroups by forming an acid ester of the hydroxyl group with adicarboxylic acid anhydride, such as succinic anhydride, or anN-dicarboximide, such as N-hydroxy succinimide. For example, thehydroxyl group of the pharmaceutically active compound can be reactedwith succinic anhydride in the presence of a base such as triethylaminein a suitable solvent such as DMF. Pharmaceutically active compoundshaving a hydroxyl group are described above. Alkanol amines are definedas including, in addition to compounds such as ethanol amine or3-propanol amine, amino acids and peptide sequences having free hydroxyland amino groups, so that alkanol amines suitable for use in the presentinvention have the structure HO--R₆ --NH₂, wherein R₆ and the preferredspecies thereof are the same as described above with respect to FormulaII.

The reaction between the copolymer and the alkanol can be performed inaqueous solution. The polymer is dissolved in the solution with anexcess, perferably at least a ten-fold excess of a alkanol amine. The pHof the solution is then adjusted to between about 4.5 and about 6 by theaddition of 0.1 N HCl1. At least a ten-fold excess of a water-solublecoupling reagent is then added with maintenance of the pH within theabove range by the addition of 1N HCl. The reaction mixture should bestirred, for about 5 to about 48 hours, acidified, and extracted into anorganic solvent such as methylene chloride, CHCl₃ dichloroethane, andthe like. The solvent extract is then washed with 1N HCl followed bywashing with saturated NaCl. The extract is then dried over anhydrousMgSO₄, filtered and concentrated to a viscous syrup. The polymer productis then precipitated using cold ether. The polymer product can then bepurified by reprecipitation from isopropanol, followed by washings withhexane and complete drying in vacuo.

Suitable water-soluble coupling reagents are well-known and disclosed bythe above-cited Bodanszky, Principles Of Peptides Synthesis, thedisclosure of which is hereby incorporated herein by reference thereto.The examples of such coupling reagents include, but are not limited to,water-soluble carbodiimides such as EDC, and3-[morpholinyl-(4)-ethyl]carbodiimide, p-toluene sulfonate,5-substituted isoxazolium salts, such as Woodward's Reagent K, and thelike.

The alkanol amide of the copolymer is then reacted with the carboxylicacid group of the pharmaceutically active compound in a solvent such asDMF, CH₂ Cl₂, pyridine, and the like. The appropriate quantities of thehydroxyl amide the polymer and the pharmaceutically active compound arecombined in the solvent, which may be heated slightly to dissolve thereactants Again, excess quantities of the pharmaceutically activecompound are preferbly employed to insure substantial conjugation of thependant hydroxyl amides of the polymer.

The reaction is carried out in the presence of one or more of thecoupling reagents and acylation catalysts described above with respectto the first mode of drug conjugation. The amount of coupling reagentand acylation catalyst should be equivalent to or in excess of theamount of pharmaceutically active compund.

The carboxylic acid group of the pharmaceutically active compound ispreferably an activated carboxylic acid group. The carboxylic acid groupof the pharmaceutically active compound can be activated by the methoddescribed above for activation of the pendant carboxylic acid group ofthe polymer. Other activating methods are well known and essentialconventional.

The reaction mixture is then stirred at between about 4 and about 40°C., and preferably about room temperature, until completion of thereaction, typically within 24 hours, usually overnight.

The hydroxyl group of the pendant alkanol amide then reacts with thecarboxylic acid group of the pharmaceutically active compound to form anester linkage. A urea product precipitates that is removed byfiltration. The product is then precipitated, filtered, dried andpurified according to the procedure described above with respect to thefirst mode of drug conjugation.

The above order of reaction may be reversed, so that the alkanol amineis first reacted with a pharmaceutically active compound having acarboxylic acid group, which carboxylic acid group may be optionallyactivated, to form a alkanol amide thereof. Suitable optional activatingsteps are well-known and essentially conventional. For example, thecarboxylic acid group of the pharmaceutically active compound can bereacted with an alkyl or p-nitrophenyl chloroformate in the presence ofa base such as triethyl amine in a suitable solvent such as DMF. Thepharmaceutically active compound with the activated carboxylic acidgroup is then precipitated, dried and purified by conventional means andreacted with the alkanol amine by the process described above for thecopolymer.

The resulting alkanol amide of the pharmaceutically active compound isthen reacted with the carboxylic acid group of the copolymer, followingthe procedure described above so that an ester linkage forms between thealkanol amide of the pharmaceutically active compound and the pendantcarboxylic acid group of the copolymer, following the proceduredescribed above for the formation of the ester linkage between thealkanol amide of the copolymer and the carboxylic acid group of thepharmaceutically active compound. The pendant carboxylic acid group ofthe copolymer is preferably activated in accordance with the optionalprocedures set forth for this mode when the alkanol amide of the pendantcarboxylic acid group of the copolymer is first formed.

As with the first mode of drug conjugation, the pendant carboxylic acidgroups of the copolymer are preferably activated pendant carboxylic acidgroups, which carboxylic acid groups can be activated in the mannerdescribed above with respect to the first mode of drug conjugation.

Drug Conjugation--Third Mode

The polymers of Formula I, having pendant carboxylic acid groups, canalso be directly conjugated with pharmaceutically active compoundshaving, prior to conjugation, carboxylic acid groups, by first reactingthe pendant carboxylic acid groups of the polymer with a diamine, sothat an amino amide of the pendant carboxylic acid group is formed. Theamino amide is then reacted with the carboxylic acid group of thepharmaceutically active compound to form an amido amide linkage betweenthe pendant carboxylic acid group of the copolymer and the carboxylicacid group of the pharmaceutically active compound. The polymers ofFormula I are described above and include polymers specificallyenumerated as preferred. Pharmaceutically active compounds having acarboxylic acid group are also described above.

Pharmaceutically active compounds having a carboxylic acid group canalso be formed from pharmaceutically active compounds having hydroxylgroups by forming an acid ester of the hydroxyl group as described abovewith respect to the second mode of drug conjugation. Pharmaceuticallyactive compounds having a hydroxyl group are also described above.Diamines are defined as including, in addition to compounds such asethylene diamine, amino acids and peptide sequences having two freeamino groups, so that diamines suitable for use with the presentinvention have the structure H₂ N--R₆ --NH₂, wherein R₆ and thepreferred species thereof are the same as described above with respectto Formula II.

The reaction between the copolymer and the diamine utilizes an aqueoussolution. The polymer is dissolved in the solution with an excess,preferably at least a ten-fold excess of the diamine, which excess isutilized in order to minimize undesirable cross linking reactions. Thependant carboxylic acid groups of the copolymer are preferably activatedpendant carboxylic acid groups, which pendant carboxylic acid groups areactivated as described above with respect to the second mode of drugconjugation.

The diamine is reacted with the pendant carboxylic acid group of thecopolymer by the same method described above with respect to the secondmode reaction between the alkanol amine and the pendant carboxylic acidgroup of the copolymer. The reaction mixture is made basic and extractedwith an organic solvent such as methylene chloride. The solvent extractis washed, dried, filtered, concentrated, precipitated and purified bythe procedure described above with respect to the alkanol amide of thecopolymer prepared pursuant to the second mode of drug conjugation.

The pendant amino amide of the copolymer is then reacted with thecarboxylic acid group of the pharmaceutically active compound asdescribed above with respect to the second mode of drug conjugation.

The carboxylic acid group of the pharmaceutically active compound ispreferably an activated carboxylic acid group. The carboxylic acid groupcan be activated by the conventional means mentioned above with respectto the second mode of drug conjugation for the reaction of the amineportion of the alkanol amine with the carboxylic acid group of thepharmaceutically active compound.

The reaction mixture is then stirred under the conditions describedabove with respect to the first mode of drug conjugation. The pendantamino amide then reacts with the carboxylic acid group of thepharmaceutically active compound to form an amide linkage. The work upand isolation of the polymer product is the same as described above withrespect to the first mode of drug conjugation.

The above order of reaction may be reversed so that the diamine is firstreacted with a pharmaceutically active compound having a carboxylic acidgroup following the procedure described above for the reaction of theamino amide of the copolymer with the carboxylic acid group of thepharmaceutically active compound. The reaction forms an amino amide ofthe carboxylic acid group of the pharmaceutically active compound. Thecarboxylic group of the pharmaceutically active compound is preferablyan activated carboxylic acid group, which may be activated byconventional means, such as by reaction with a carbodiimide.

The resulting amino amide of the carboxylic acid group of thepharmaceutically active compound is then reacted with the pendantcarboxylic acid group of the copolymer following the procedure describedabove for the reaction of the diamide with the pendant carboxylic acidgroup of the copolymer. An amide linkage is formed between the aminoamide and the pendant carboxylic acid group. The pendant carboxylic acidgroup is preferably an activated carboxylic acid group, prepared asdescribed above with respect to the first mode of drug conjugation.

Drug Conjugation--Fourth Mode

The polymers of Formula I, having pendant carboxylic acid groups, canalso be conjugated with pharmaceutically active compounds that, prior toconjugation, have an aldehyde, ketone or carboxylic acid group. Thefourth mode of drug conjugation first forms pendant acyl hydrazinegroups from the pendant carboxylic acid groups of the polymer, whichacyl hydrazine is then reacted with the aldehyde, ketone or carboxylicacid group of the pharmaceutically active compound to form a hydrazoneor diacyl hydrazide linkage between the copolymer and thepharmaceutically active compound. The polymers of Formula I aredescribed above and include polymers specifically enumerated aspreferred. Pharmaceutically active compounds having an aldehyde, ketoneor carboxylic acid group are also described above.

Pharmaceutically active compounds having a carboxylic acid group canalso be formed from pharmaceutically active compounds having hydroxylgroups by forming an acid ester of the hydroxyl group as described abovewith respect to the second mode of drug conjugation. Pharmaceuticallyactive compounds having a hydroxyl group are also described above.

The fourth mode of drug conjugation first forms pendant acyl hydrazinegroups on the polymer by reacting the pendant carboxylic acid groups ofthe polymer with an alkyl carbazate, so that the pendant carboxylic acidgroups form pendant alkyl carbazate groups. The alkyl portion is actingas a protecting group. It is removed in the subsequent step to yieldacyl hydrazine. The reaction utilizes an organic solvent such asmethanol in which the polymer and an excess, preferably at least aten-fold excess of an alkyl carbazate are reacted at between about 4° C.and about 40° C. in the presence of an excess quantity of a couplingreagent. The most preferred alkyl carbazate is t-butyl carbazate.Examples of suitable coupling reagents include those listed above withrespect to the first mode of drug conjugation. Likewise, the work-up andisolation of the polymer product is the same as described above withrespect to the first mode of drug conjugation.

The alkyl carbazate group is then removed to form pendant acyl hydrazinegroups by mixing the polymer with a 4M solution of HCl in dioxane. Themixture is stirred for between about 30 min. and about 2 hours at roomtemperature, with the polymer settling at the bottom as an oil. Thehydrochloride salt of the hydrazine is then worked-up and isolated asdescribed above.

The polymer having pendant acyl hydrazine groups is then conjugated withthe pharmaceutically active compound. The conjugation reaction utilizesan organic solvent in which the reactants are soluble. Examples ofsuitable organic solvents include pyridine, DMF, CH₂ Cl₂, THF, and thelike. The polymer having pendant acyl hydrazine groups and thepharmaceutically active compound are dissolved in the solvent andreacted as disclosed above with respect to the first mode of drugconjugation.

The pendent acyl hydrazine groups of the polymer react with the aldehydeand ketone to form a hydrazaone or with the carboxylic acid group of thepharmaceutically active compound to form a diacyl hydrazide linkage.Hydrazaones can be formed with aldehyde or ketone containing drugs(adriamycine, testosterone) or when aldehydes or ketones are introduced(e.g., by oxidation of carbohydrate residues of glycopeptides such asdisclosed by the co-pending U.S. patent application Ser. No. 673,696 byZalipsky et als, filed Mar. 15, 1991, the disclosure of which is herebyincorporated herein by reference thereto). The work-up and isolation ofthe polymer product is the same as described above with respect to thefirst mode of drug conjugation.

When the pharmaceutically active compound has a carboxylic acid group,the carboxylic acid group is preferably an activated carboxylic acidgroup, substituted with a suitable leaving group capable of beingdisplaced by the pendant acyl hydrazine group of the polymer. Examplesof suitable leaving groups are disclosed by Bodanszky, Principals ofPeptide Synthesis, cited above, the disclosure of which is herebyincorporated herein by reference thereto. Such leaving groups, which arewell-known, include, but are not limited to, imidazolyl, triazolyl,N-hydroxy succinimidyl, N-hydroxy norbornene dicarboximidyl and phenolicleaving groups, and are substituted onto the carboxylic acid group ofthe pharmaceutically active compound by reacting the carboxylic acidgroup in the presence of an activating reagent with the correspondingimidazole, triazole, N-hydroxy succinimide, N-hydroxy norbornenedicarboximide and phenolic compounds. Suitable activating reagentsinclude those disclosed above with respect to the first mode of drugconjugation.

Drug Conjugation--Fifth Mode

The polymers of Formula III having pendant amino groups can be directlyconjugated with pharmaceutically active compounds that, prior toconjugation, have a carboxylic acid group. The polymers of Formula IIIare described above, and include polymers specifically enumerated aspreferred. Pharmaceutically active compounds having carboxylic acidgroups are also described above. Pharmaceutically active compoundshaving a carboxylic acid group can also be formed from pharmaceuticallyactive compounds having hydroxyl groups by reacting the hydroxyl groupas described above with respect to the second mode of drug conjugation.Pharmaceutically active compounds having a hydroxyl group are describedabove.

The polymer and the pharmaceutically active compound are reacted andrecovered as described above in the fourth mode of drug conjugation forthe reaction between the polymer having pendant acyl hydrazine groupsand the pharmaceutically active compound having carboxylic acid groups.The pendant amino group of the polymer reacts with the carboxylic acidgroup of the pharmaceutically active compound to form an amide linkage.

The carboxylic acid group of the pharmaceutically active compound ispreferably an activated carboxylic acid group, substituted with asuitable leaving group capable of being displaced by the pendant aminogroup of the polymer. The activation of such carboxylic acid groups iswell-known and essentially conventional. The carboxylic acid groups ofthe pharmaceutically active compounds can be activated as describedabove with respect to the fourth mode of drug conjugation.

The polymers of Formulas I and III can also be conjugated withbiologically active polypeptides and glycopolypeptides. Biologicallyactive polypeptides and glycopolypeptides of interest include thoselisted in the above-incorporated con,ending U.S. patent application Ser.No. 627,696, now abandoned by Zalipsky et als. The biologically activepolypeptides and glycopolypeptides contain aldehyde, ketone andcarboxylic acid groups that can be conjugated with the polymers of thepresent invention according to the third, fourth and fifth modes of drugconjugation, or according to the methods described in theabove-incorporated U.S. patent application Ser. No. 672,696, nowabandoned by Zalipsky et als.

Targeted Immunotherapy

Another class of biologically active glycopolypeptides of interest aremonoclonal antibodies. Monoclonal antibodies contain carbohydratemoieties capable of being oxidized to form aldehydes and ketones. Thegroups can be generated on the carbohydrate moieties, for example, byoxidizing the vicinal diols of the carbohydrate moieties with excessperiodate, or enzymatically, e.g. by use of galactose oxidase, using themethods described in the above-incorporated U.S. patent application Ser.No. 673,696 by Zalipsky et als.

Clearly, the ketones and aldehydes of the oxidized carbohydrate moietiesof monoclonal antibodies can be coupled with the polymers of Formula Iby the fourth mode of drug conjugation disclosed above. Sodiumborohydride or sodium cyanoborohydrate is added to the reaction mixtureto reduce the resulting hydrazone to a more stable alkyl hydrazide.

However, the oxidized carbohydrate moieties of monoclonal antibodieswill also react with amino amides formed from pendant carboxylic acidgroups of the polymers of Formula I, according to the third mode of drugconjugation, as well as with the pendant amino groups of the polymers ofFormula III, according to the fifth mode of drug conjugation, in thepresence of sodium borohydride. The attachment of a single monoclonalantibody to a polymer is sufficient to bind the polymers to cells forwhich the monoclonal antibody is specific.

When a polymer according to Formula I or Formula III has monoclonalantibodies conjugated thereto, the polymer can be co-conjugated with apharmaceutically active compound to deliver the compound to the specificcell the monoclonal antibodies function to bind the polymer to. Specificcells can be targeted for treatment by the pharmaceutically activecompound, significant quantities of which will not be delivered to othertissues. This is particularly important in applications when thepharmaceutically active compound produces toxic or other undesirableside effects in tissues not intended for treatment. Lower dosagequantities will also be possible because application of thepharmaceutically active compound will be essentially limited to thetreatment site. For example, chemotherapeutic compounds can be used totreat cancerous cells that would otherwise be toxic to healthy tissues.

The co-conjugates of pharmaceutically active compounds and monoclonalantibodies with the polymers of Formulas I and III are formed by firstreacting the pharmaceutically active compound with the polymer accordingto either the third, fourth or fifth mode of drug conjugation. An excessof polymer is utilized so that pendant functional groups will remainunconjugated for the attachment of the monoclonal antibody. Thecarbohydrate moieties of the monoclonal antibody are first oxidized toproduce aldehyde and ketone groups for conjugation, and the monoclonalantibody is then reacted with the conjugate of the pharmaceuticallyactive compound and the copolymer of Formula I or III having availablependant functional groups according to the third, fourth or fifth modeof drug conjugation, as if the monoclonal antibody were apharmaceutically active compound. As noted above, the reaction can beperformed in the presence of sodium borohydride or sodiumcyanoborohydrate to convert the resulting hydrazone to a hydrazide. Theco-conjugates of the pharmaceutically active compound and monoclonalantibody with the polymer of Formula I or III can then be purified byprotein chromatography by conventional methods.

A number of useful combinations of pharmaceutically active compounds andmonoclonal antibodies are available for the treatment of specific celltypes in need thereof with suitable pharmaceutically active compounds.For example, as described above, chlorin e₆, a photosensitizer, can beco-conjugated with an anti-T cell monoclonal antibody to bind thepolymer-drug conjugation to T-cell leukemia cells. Thus, only theT-cells are rendered photosesitive and subsequent treatment withultraviolet light substantially reduces or eliminates the T-cellleukemia cells without affecting other types of cells.

Other pharmaceutically active compounds preferably co-conjugated withmonclonal antibodies include cytotoxic drugs such as daunomycin,metotrexate, cytorhodin-S, adriamycin, mitomycin, doxorubicin, melphalanand the like. Metal chelating compounds such as EDTA can beco-conjugated with monoclonal antibodies to form complexes withradioative isotpes for the treatment of cells in need thereof, to whichthe monoclonal antibody is capable of binding. Examples of radioactiveisotopes include, but are not limited to ⁹⁹ Tc and ¹²³ I, which can beused for example in the treatment of cancerous cells.

A large number of pharmaceutically active compounds may be conjugatedwith the polymers of Formulas I and III, including antibiotics,anti-neoplastic agents, antiviral agents, cytotoxic drugs, metalchelators, hormones, and the like. The resulting conjugate can beprepared for administration by incorporating the same into a suitablepharmaceutical formulation.

Examples of suitable pharmaceutical formulations are well-known in theart and may include, but are not limited to, phosphate buffered salinesolutions, water, emulsions such as oil/water emulsion, and varioustypes of wetting agents. Other suitable pharmaceutical formulationsinclude sterile solutions, tablets, coated tablets and capsules.

Typically, such pharmaceutic formulations contain excipients such asstarch, milk, sugar, certain types of clay, gelatin, stearic acid orsalts thereof, such as magnesium or calcium stearate, talc, vegetablefats or oils, gums, glycols, and the like. Such formulations may alsoinclude flavor and color additives or other ingredients. Compositions ofsuch formulations are prepared by well-known conventional methods.

The invention also provides a method for treating a pathologicalcondition in a subject in need thereof by administering to the subjectthe composition of the present invention. Administration of themedication may occur in one of several ways, including oral, intravenus,intraperitoneal, subcutaneous, intramuscular, topical or intradermaladministration.

Ionically Conductive Materials

Certain of the polymers of Formula I form an ionically conductivematerial when combined with an alkali metal electrolyte salt. Thepolymers of Formula I are described above, and include polymersspecifically enumerated as preferred. The polymers of Formula I capableof forming ionically conductive materials are those polymers in which Yis --OH or a C-terminus protecting group having the structure --OR₇,wherein R₇ is an alkyl group and preferably an ethyl group.

The alkali metal electrolyte salt is preferably a lithium electrolytesalt. Suitable lithium salts include LiAsF₆, LipF₆, LiI, LiBr, LiBF₆,LiAlCl₄, LiCF₃ CO₂, LiCF₃ SO₃. Preferred lithium electrolyte saltsinclude LiAsF₆, LipF₆, LiI and LiCF₃ SO₃. The most preferred lithiumelectrolyte salts are LiAsF₆ and LiCF₃ SO₃.

The preparation of the ionically conductive materials utilizes anorganic solvent in which the polymer and the alkali metal electrolytesalt are soluble, such as acetonitrile. The ratio of polymer toelectrolyte salt should be between about 2:1 and about 10:1 andpreferably about 4:1. The total solution concentration (w/v %) of bothcompounds combined is between about 1 percent and about 25 percent, andpreferably about 10 percent, depending upon the solubility of thematerials. The polymer and electrolyte salt are dissolved in thesolvent, which may be heated slightly to dissolve the materials. Themixture is cast into the desired form, and the solvent is removed bydrying, first in air and then under vacuum. The mixture may be heated toremove the solvent.

The polymers preferably have a molecular weight greater than about75,000 daltons to provide the mixture with adequate mechanical strength.The polymers are also preferably cross-linked, as set forth below, toprovide adequate mechanical strengths to the material. The mixture ofpolymer and electrolyte salt with solvent removed may also becompression molded to obtain articles having a desired form.

The ionically conductive materials of the present invention are usefulas electrodes in electrochemical cells. However, the ionicallyconductive materials of the present invention, instead of being utilizedas electrodes, are particularly useful as solid electrolytes fornon-aqueous electrochemical cells. The mixture is particularly wellsuited for use in non-aqueous secondary cells.

Non-aqueous electrochemical cells can be assembled utilizing theionically conductive material of the present invention by combining acathode, an anode and a solid electrolyte containing the ionicallyconductive material. Examples of suitable anodes include alkali metalssuch as sodium, potassium and lithium. The alkali metal electrolyte saltwould then be a salt of the metal utilized. The preferred alkali metalis lithium.

The anode can also be a counter-electrode capable of reversiblyintercalating lithium from the cathode. In this instance, then, thealkali metal electrolyte salt must be a lithium salt. Anodes thatfunction as counter-electrodes capable of reversibly intercalatinglithium are well-known and are prepared from graphitic carbon.

The cathode preferably contains a cathode-active material capable ofreversibly intercalating lithium. Suitable lithium-intercalable cathodematerials include metal-chalcogen combinations, particularly transitionmetal-chalcogen combinations, metal halides, and the like. Chalcogensare understood by those of ordinary skill in the art to include thechemically-related elements from Group VI of the periodic table, namelyoxygen, sulfur, selenium, tellurium and polonium. The preferredchalcogens are oxygen and sulfur. Preferred transition metals includemanganese, nickel, iron, chromium, titanium, vanadium, molybdenum andcobalt. Preferred compositions include molybdenum sulfides, vanadiumoxides and manganese oxides. MoS₂, V₆ O₁₃, Mo₆ S₈ and MnO₂ are morepreferred, with MnO₂ being most preferred.

It is desirable that the cathode, as well as the anode, whencarbonaceous materials are utilized, maintain their electricalconductivity at all states of charge. Conductivity may be enhanced byutilizing the ionically conductive materials of the present invention asa binder for the cathode-active materials and for the carbonaceouscounter-electrodes of the anode.

In assembling the cells of the present invention, the cathode istypically fabricated by depositing a slurry of a cathode-activematerial, ionically conductive binder and a fugitive liquid carrier suchas one of the solvents utilized in the preparation of the ionicallyconductive materials, on a cathode current collector, and thenevaporating the carrier to leave a coherent mass in electrical contactwith the current collector. Likewise, the anode may be prepared bydepositing a slurry of a carbonaceous anode material, the ionicallyconductive binder and the fugitive liquid carrier on anelectrically-conductive anode support and then evaporating the carrierto leave a coherent mass in electrical contact with the anode support.The cell is then assembled by sandwiching the cathode and anode layerswith the solid electrolyte containing the ionically conductive materialof the present invention layered therebetween. The anode and cathodecurrent collectors are then placed in electrical contact with theirrespective anode and cathode terminals. The ionically conductive bindermay be present in an amount between about 0.5 percent and about 25percent by weight of the cathode or anode material, and preferablybetween about 2 percent and about 10 percent by weight.

Cross-Linked Polymer Products

The polymers of Formulas I and III can also be cross-linked to formpolymer matrices that can be utilized in the preparation of hydrogelmembranes and semi-interpenetrating polymer networks (semi-IPN's). Thepolymers of Formulas I and III can be cross-linked by way ofhydrolytically stable urethane linkages between a trifunctional amineand the poly(alkylene oxide) moiety of the copolymer. The polymers ofFormula I, having pendant acyl hydrazine groups, can also becross-linked by way of hydrolytically labile acyl semicarbazide linkagesbetween a diisocyanate and the pendant acyl hydrazine groups of thepolymer. The cross-link density of the polymer matrix can be controlledby varying the length of the poly(alkylene oxide) moiety of the polymersof Formulas I and III. The polymers of Formulas I and III are describedabove and include polymers specifically enumerated as preferred. Thepolymer matrices cross-linked by way of acyl semicarbazide linkagesutilize polymers according to Formula I having pendant acyl hydrazinefunctional groups that are prepared as described above with respect tothe fourth mode of drug conjugation.

As noted above, the cross-linking reaction to form urethane linkagesreacts a trifunctional amine with the poly(alkylene oxide) moiety of thecopolymer. Accordingly, polymers having terminal poly(alkylene oxide)groups should be used. Such polymers can be obtained from thepolymerization processes of the present invention by reacting the aminoacids or peptide sequences with an excess of poly(alkylene oxide).

The terminal poly(alkylene oxide) groups should be activatedpoly(alkylene oxide) groups. The polymers of Formula I produced by theinterfacial polymerization process described above will have activatedterminal poly(alkylene oxide) groups. The polymers of Formula III areprepared by the solution polymerization processes described above, whichdo not result in polymers having activated terminal poly(alkylene oxide)groups. However, the terminal poly(alkylene oxide) groups of thepolymers of Formula III can be activated by the methods described abovewith respect to the interfacial polymerization process for thepreparation of the polymers of Formula I. However, the activation stepshould not be performed until after the polymerization of the polymersof Formula III.

The urethane cross-linking reaction utilizes an solvent in which thereactants are soluble. Examples of suitable solvents include methylenechloride, chloroform, THF, dioxane, water, DMF, acetonitrile, and thelike. Equivalent quantities of the polymer and the trifunctional amineare reacted. Trifunctional amines are defined as any compound havingthree free amine groups, including aromatic materials. Suitabletrifunctional amines include any soluble material having three aminesthat can be used as a cross-linking agent. preferred trifunctionalamines have the structure N(--R₆ --NH₂)₃, in which R₆ is the same asdescribed above with respect to Formula II. Trifunctional amines, thealkyl moieties of which have between about 1 and about 10 carbon atomsare preferred. Trifunctional amines with alkyl moieties having betweenabout 2 and about 6 carbon atoms are even more preferred.

Separate solutions of the polymer and the trifunctional amine areprepared. The solvents may be heated slightly to dissolve the reactants.The solution concentration (w/v %) of the polymer solution should beless than about 10 percent so that cross-linking of the polymer does notoccur too rapidly.

The solution of the amine is added to the polymer solution withstirring. Within minutes the mixture is poured into molds, with thesolvent permitted to slowly evaporate. The evaporation is usuallycomplete within 24 hours, typically overnight. The cross-linked polymermatrix forms a film that can be peeled from the mold. N-hydroxysuccinimide is a bi-product of the cross-linking reaction and willremain embedded in the polymer matrix unless removed by washing withwater. However, this is readily accomplished by rinsing the membranewith several successive washings of distilled, deionized water. Analysisof the washing has shown that substantially all of the N-hydroxysuccinimide is removed by the first washing.

Polymer matrices cross-linked by urethane linkages can also be preparedutilizing the poly(alkylene oxide) homopolymers disclosed above asstarting materials for the interfacial polymerization process describedabove. In other words, it is not necessary for this cross-linking methodthat the poly(alkylene oxide) be copolymerized with an amino acid orpeptide sequence.

The acyl semicarbazide cross-linking of the polymers of Formulas Ihaving pendant acyl hydrazine groups does not require the use ofpolymers having terminal alkylene oxide moieties. The reaction utilizesthe same organic solvents utilized in the urethane cross-linkingreaction. Equivalent quantities of the polymer and the diisocyanate arereacted. Suitable diisocyanates have the structure O═C═N--R₆ --N═C═O, inwhich R₆ is the same as described above with respect to Formula II.Alkyl diisocyanates, the alkyl moieties of which have between 1 andabout 10 carbon atoms are preferred. Alkyl diisocyanates with alkylmoieties having between about 2 and about 6 carbon atoms are even morepreferred. Aromatic diisocyanaters such as toluene diisocyanate are alsosuitable for use with the present invention. The solution concentration(w/v %) of the polymer should again be less than 10 percent so thatcross-linking does not occur too rapidly.

The polymer is dissolved first and the solvent may be heated slightly todissolve the material. To this solution is added an excess of a basesuch as triethylamine or sodium bicarbonate to convert the hydrochlorideof the pendant hydrazine groups to the free base. Once the conversion iscomplete, the solution is filtered and the residue washed with thereaction solvent. The alkyl diisocyanate is added to the combinedfiltrate with stirring. Within minutes the solution is poured intomolds, with the solvent permitted to slowly evaporate. Evaporation isusually complete within 24 hours, typically overnight. The cross-linkedpolymer matrix forms a film that can be peeled from the mold.

The above-disclosed diisocyanate can be substituted with otherbifunctional compounds. Examples of suitable bifunctional compoundsinclude diglycidyl ethers, dialdehydes such as glutaraldehyde, aliphaticand aromatic dicyanates such as Bisphenol A dicyanate, and diamines suchas ethylene diamine or hexamethylene diamine.

Both the polymer matrices cross-linked with urethane linkages and thepolymer matrices cross-linked with acyl semicarbazide linkagesdemonstrate high equilibrium water content and good mechanical strengthand are therefore suitable for biomedical applications such as wounddressings and implant materials. The hydrogel membranes from both typesof cross-linked linkages are translucent and flexible films in the drystate. The urethane cross-linked membranes are generally more opaque andsomewhat abrasive on the surface, from the presence of N-hydroxysuccinimide liberated during the cross-linking reaction. In the drystate, the membranes have extremely high tensile strength andelongation.

When equilibrated with water, the membranes begin to swell almostinstantaneously, with the equilibrium reached in less than one hour. Themembranes are elastic in the swollen state, with tensile strengthindependent of the molecular weight of the poly(alkylene oxide) used.

The mechanical properties of the polymer matrices can be furtherimproved by forming semi-IPN's with the matrices. A linear, preformedsecond polymer is entrapped within the polymer matrices, which secondpolymer is chosen to be biocompatible and to contribute to themechanical properties of the polymer matrix. The second polymer need notbe miscible with the polymers of the present invention. Stated anotherway, the semi-IPN's of the present invention can be formed from polymersthat would not be physically blendable by any other means. Examples ofsecond polymers suitable for use with the semi-IPN's of the presentinvention include poly(BPA carbonate), poly(desaminotyrosyl tyrosinehexyl ester carbonate), poly(lactic acid), poly(caprolactone), celluloseacetate, cellulose nitrate, poly(ethylene terephthalate) poly(styrene)and poly(methyl methacrylate), and the like.

Semi-IPN's can be prepared by either cross-linking reaction with boththe polymers of Formulas I and III. The semi-IPN's are prepared bydissolving an equimolar amount of the second polymer in the organicsolvent with the polymer of Formula I or III. The reaction then proceedsas described above, with respect to the preparation of polymer matricescross-linked by either urethane or acyl semicarbazide linkages. Thesecond polymer is then entrapped within the cross-linked polymer matrix,as the polymer matrix is formed.

Both the cross-linked polymer matrices and the semi-IPN's can be used asmeans for drug delivery when utilized as wound dressings or biomedicalimplants. The polymer matrices cross-linked by urethane linkages fromthe polymers of Formulas I and III are not cross-linked by means oftheir pendant functional groups, which remain available for drugattachment. The trifunctional amine can also be quaternized for theattachment of pharmaceutically active compounds. While the polymermatrices that are cross-linked by acyl semicarbazide linkages covalentlybond with the diisocyanate by means of their pendant functional groups,not all pendant functional groups participate in the cross-linking, andan excess of polymer can also be utilized, so that pendant functionalgroups remain uncross-linked for drug attachment. Wound dressingsprepared from hydrogel membranes or semi-IPN's of the polymer matricescan thus incorporate antibiotics to promote wound healing.

In view of the foregoing, it can be readily appreciated that thepoly(alkylene oxide) copolymers of the present invention are versatiledrug carriers derived from biocompatible components that are capable ofbeing adapted to conjugate with a number of drug functional groups, soas not to be limited by drug structure or activity. The drug carrierscan be administered in a variety of forms that are dominated by thedesirable properties of the poly(alkylene oxides) from which thecarriers are derived.

The following non-limiting examples set forth hereinbelow illustratecertain aspects of the invention. All parts and percentages are byweight unless otherwise noted, and all temperatures are in degreesCelsius.

EXAMPLES Example 1

Preparation of PEG-bis Succinimidyl-Carbonate

The preparation of PEG-his Succinimidyl Carbonate is disclosed in theabove-incorporated U.S. patent application Ser. No. 340,928 by Zalipsky.In a 250 mL round-bottomed flask, 10 g (10 mmols of hydroxyl groups) ofPEG 2000 (Fluka) was dissolved in 120 mL of toluene and the polymersolution was azeotropically dried for two hours under reflux using aDean-Stark trap. The polymer solution was then cooled to 25° C. and 15mL (29 mmol) of a 20 percent solution of phosgene in toluene (1.93M) wasadded. The reaction mixture was stirred at 25° C. overnight and thenevaporated to dryness on a rotary evaporator (water bath temperaturemaintained at 40° C.). Another 100 mL of toluene was added andevaporated to remove all traces of phosgene. To the polymericchloroformate was added 30 mL of dry toluene, 10 mL of methylenechloride, and 1.7 g (14.8 mmol) of N-hydroxy succinimide, and themixture was stirred vigorously. The reaction flask was then cooled in anice water bath and 1.5 g (14.9 mmol) of triethylamine was addedgradually. Immediate precipitation of triethylamine hydrochloride wasseen. The cooling bath was removed and the stirring continued at 25° C.for five hours. Then 10 mL of toluene was added and the reaction mixturecooled to 4° C. to maximize the triethylamine hydrochlorideprecipitation.

The precipitate was filtered and the filtrate concentrated to about halfof its original volume. The concentrated solution was then added to 60mL of ether with stirring to precipitate the polymeric product. Aftercooling to 40° C., the crude product was recovered by filtration, dried,redissolved in 100 mL of 2-propanol at 45° C. and allowed torecrystallize. The product was recovered by filtration, washed withether and dried under high vacuum. The recovery of the white crystal andsolid was 74 percent.

Example 2

Preparation of PEG-Lysine Ethyl Ester copolymer (Poly(PEG-Lys--OEt)

In a 500 mL three-necked round-bottomed flask fitted with an overheadstirrer was dissolved 1.1 g (4.4 mmol. of lysine ether esterhydrochloride salt (Fluka) and 1.7 g (21 mmol) of sodium bicarbonate in100 mL of water. The PEG-N-hydroxy succinimide-dicarbonate of Example 1(10 g, 4.4 meq) was dissolved in 200 mL of methylene chloride and addedto the reaction mixture. The mixture was stirred vigorously (about 1100rpm) for two hours and then acidified to about pH 2. The two phases wereseparated and the organic phase was washed twice with NaCl. The organiclayer was then dried over anhydrous MgSO₄, filtered and concentrated.The polymer was precipitated using cold ether, cooled to 4° C. andfiltered to recover 6.7 g (67 percent) of the polymer.

500 mg of the crude polymer was dissolved in 10 mL of distilled waterand dialyzed against distilled water at room temperature for 48 hoursusing a SPECTRAPOR™ membrane with a molecular weight cut-off of 12,000to 14,000 daltons. The purified polymer was extracted with methylenechloride, washed with saturated NaCl solution, dried and evaporated toobtain 263 mg (53 percent) of pure polymer.

Example 3

Preparation of pEG-Lysine Copolymer (Poly(PEG-Lys)

5 g of the polymer of Example 2 was dissolved in 5 mL of H₂ O. The pH ofthe polymer solution was about 5 as measured with a pH meter. A 0.01NNaOH solution was prepared, and the base was added dropwise into thepolymer solution with stirring. The pH was monitored continuously andkept around 11.5 by the addition of base as needed. The reaction wasallowed to go for five hours. The reaction was stopped and the reactionmixture was acidified with 0.1N HCl. The polymer was extracted intomethylene chloride and the extract was washed with saturated NaCl, driedover anhydrous MgSO₄, filtered and concentrated. The polymer was thenprecipitated with cold ether. After cooling for several hours, theproduct was collected in a Buchner funnel, washed with cold ether anddried under vacuum overnight. 3.5 g of polymer (71 percent) wasrecovered.

Example 4

Preparation of Activated Poly(PEG,Lys)

In a 10 mL round-bottomed flask, 1.0 g (0.46 mmol) of the polymer ofExample 3 was dissolved in 5 mL of methylene chloride. To this solution0.26 g of N-hydroxy succinimide (Aldrich) (2.3 mmol) was added. Theflask was cooled in an ice water bath and 0.10 g (0.50 mmol) ofdicyclohexyl carbodiimide (DCC) (Aldrich) was added. The reactionmixture was then stirred at 0° C. for one hour and at room temperatureovernight. The reaction mixture was filtered to remove dicyclohexyl ureaand the methylene chlorine was evaporated to give a white, waxymaterial. To this 5 mL of isopropanol was added and the mixture wasstirred until a clear solution was obtained. Cooling to -15° C.precipitated a white solid which was collected on a Buchner funnel andwashed first with isopropanol and then with hexane. The material wasfurther purified by recrystallization from isopropanol. The recovery ofthe final product was 0.72 g (71 percent).

Example 5

Preparation of Poly(PEG-Lys) with Pendant Acyl Hydrazine FunctionalGroups

In a 50 mL round-bottomed flask, 2.2 g (1.0 mmol) of the polymer ofExample 3 was dissolved in 20 mL of methylene chloride. The flask wasthen cooled in an ice water bath. To the flask were added 410 mg (2.0mmol) of DCC and 260 mg (2.0 mmol) of tert-butyl carbazate (Aldrich).The contents of the flask were stirred at ice water bath temperature for1 hour and then stirred at room temperature for 24 hours. The reactionmixture was filtered to remove the dicyclohexyl urea, followed byevaporation of the filtrate to dryness, which gave 1.5 g of light solidthat was purified by recrystallization from 2-propanol. ¹ H proton NMRspectrum of the white, waxy solid showed tert-butyl peaks, the area ofwhich corresponded to greater than 90 percent conversion. Whenredissolved in methanol and reprecipitated with ether, the relativeintensity of this peak did not decrease.

An approximate 4M solution of HCl in dioxane was prepared by bubblingHCl gas through dioxane in an Erlenmeyer flask (a 4.0M solution is alsoavailable commercially from (Pierce). In a 250 mL round-bottomed flaskwas placed 75 mL of the 4.0M HCl dioxane solution, and to this was addedwith stirring 5.0 g of the polymer-carbazate reaction product in theform of small pieces. Stirring was continued for two hours at roomtemperature. The polymer settled at the bottom of the flask as an oil.The dioxane/HCl layer was decanted out and the polymer layer was addedto 100 mL of the ether with stirring. The polymer precipitated and wasisolated, washed twice with 50 mL of ether and dried under vacuum. Itwas further precipitated by recrystallization from isopropanol.

The ¹ H NMR spectrum of the product showed the complete absence oftert-butyl groups. Non-aqueous titration against sodium methoxide withmethyl red as the indicator showed about 100 percent of the expectedhydrochloride.

Example 6

Preparation of Poly(PEG-Lys) Having Ethanol Pendant Functional Groups

In a 50 mL round-bottomed flask, 0.400 g (0.1819 mmol) of thepoly(PEG-Lys) of Example 3 was dissolved in 40 mL of water. To thissolution was added 0.1 mL (1.656 mmol) of ethanol amine (Aldrich). ThepH was adjusted to 4.75 by the addition of 0.1N HCl. Then 0.348 g (1.82mmol) of solid 1-(3-dimethylaminopropyl-3-ethylcarbodiimide) (Sigma) wasadded. The pH had a tendency to increase, but was maintained around 4.75by the addition of 1N HCl. After 30 minutes, no further increase in pHwas observed. The reaction mixture was stirred overnight and thenacidified and extracted into methylene chloride. The methylene chlorideextract was washed with saturated sodium chloride solution, dried withanhydrous magnesium sulfate, filtered, concentrated to a viscous syrupand precipitated with cold ether. About 0.318 g of crude poly(PEG-Lys)with ethanol amide pendant functional groups was recovered. The crudeproduct was purified by reprecipitation from isopropanol, followed bywashings with hexane and complete drying in vacuo. Thin layerchromatography (TLC) in a 4:1 ratio solution of ethanol to ammoniashowed an absence of free ethanol amine.

Example 7

Preparation of Poly(PEG-Lys) Having Ethylamine Pendant Functional Groups

In a 100 mL three-necked flask, 1.21 g (0.55 mmol) of the poly(PEG-Lys)of Example 3 was dissolved in 80 mL of water. To this solution was added0.37 mL (5.5 mmol) of ethylene diamine (Aldrich). The pH was adjusted to4.75 by the addition of 1N HCl. Then 1.05 g (5.5 mmol) of solid1-(3-dimethylaminopropyl-3-ethylcarbodiimide) was added. The pH had atendency to increase, but was maintained around 4.75 by the addition of1N HCl. After 30 minutes, no further increase in pH was observed. Thereaction mixture was stirred overnight and then made basic and extractedinto methylene chloride. The methylene chloride extract was washed withsaturated sodium chloride, dried with anhydrous magnesium sulfate,filtered, concentrated to a viscous syrup and precipitated with coldether. About 0.725 g of crude poly(PEG-Lys) having ethylamine pendantfunctional groups was recovered, which was purified by reprecipitationwith isopropanol. TLC in a 2:1 solution of ethanol to ammonia showedabsence of free diamine.

Example 8

Preparation of Poly(PEG-Lys) having Pendant Hexylamine Functional Groups

The procedure of Example 7 was followed substituting 5.5 mmol ofhexamethylene diamine (Aldrich) for the 5.5 mmol of the ethylenediamine. Upon purification of the product, TLC in a 2:1 ratio ethanol toammonia solution showed absence of free diamine.

Example 9

Preparation of Poly(PEG-Lys)-Cephradine Drug conjugate

In a 25 mL round-bottomed flask, 0.1523 g of cephradine (0.436 mmoles)(Sigma) was dissolved in a mixture of 4.5 mL water and 2 mL of dioxane.To this solution, 0.500 g of the activated poly(PEG-Lys) of Example 4(0.218 mmoles) was added. This was followed by the addition of 0.055 gof NaHCO₃. The solution was stirred at room temperature. The pH of thereaction was monitored and was found to remain in the narrow range fromabout 7.0 to 7.5. The reaction mixture was neutralized after one hour byadding a few drops at 0.1N HCl and extracted into methylene chloride.The extract was washed with saturated sodium chloride, dried overanhydrous magnesium sulfate, filtered and concentrated. The polymer wasthen precipitated with cold ether. After cooling for several hours, theproduct was collected on a Buchner funnel, washed with cold ether anddried under vacuum overnight. The recovery was 0.355 g, or 71 percent.

The reaction product was then dissolved in water (50 mg/mL) and dialyzedagainst distilled water at room temperature using a SPECTRAPOR™ membranehaving a molecular weight cutoff of 12,000 to 14,000 daltons. After 24hours the product was isolated by lyophilization.

Examples 10-12

Attachment of Cephradine to Poly(PEG-Lys); Optimization of Conditions

Example 9 was repeated at pH's of 7.2 and 8.5, attachment was_(I)determined by iodometric assay, which reaction times of 1.5 and 3 hours,and polymer to drug ratios of 1:1. The mole-percent degree of drugmethod measures only the active drug. As shown in Table I, the greatestdegree of drug attachment was obtained with the conditions of Example 9,namely, a reaction time of 1 hour, a pH of 7.5 and a ratio of polymer todrug of 1:2.

                  TABLE I                                                         ______________________________________                                                                 Polymer: Drug                                                                           Degree of                                  Example   Time   pH      Ratio     Attachment.sup.1                           ______________________________________                                         9        1      7.5     1:2       61.5                                       10        3      8.5     1:1       41.5                                       11        1.5    8.5     1:1       40                                         12        1.5    7.2     1:1       50                                         ______________________________________                                         .sup.1 Mole %                                                            

Table I shows that decreasing the reaction time from 3 to 1.5 hours hadno significant effect on the degree of drug attachment. This is expectedbecause the active ester would not be stable under the conditions of thereaction for a long period of time. Also, the amount of active drug onthe polymer is higher when the reaction is done at a lower pH. Becausethe iodometric assay is specific for active drug, this could mean thatat a pH of 8.5 some of the beta-lactam units of the drug may have beenhydrolyzed. Thus, the optimum reaction conditions appear to be mildenough to prevent significant cleavage of the beta-lactam ring while atthe same time giving a high degree of conjugation.

Example 13

Preparation of a Poly(PEG-Lys)-Penicillin V Drug Conjugate

In a 10 mL round-bottomed flask, 0.400 g (0.178 mmol) of thepoly(PEG-Lys) having ethanolamide pendant functional groups of Example 6was dissolved in 4 mL of methylene chloride. To this solution was added0.094 g (0.267 mmol) of Penicillin V (Sigma) and 0.008 g (0.065 mmol) ofdimethylaminopyridine (Aldrich). The reaction mixture was cooled in anice water bath and then 0.048 g (0.232 mmol) of DCC was added. After afew hours at 0° C., the reaction vessel was moved to a cold roommaintained at 4° C. and allowed to stir for almost six days. Aprecipitate of dicyclohexyl urea formed and was removed by filtration.The drug conjugate was precipitated with cold ether. About 0.250 g ofcrude product was obtained which was purified by reprecipitation twicefrom isopropanol. TLC in methanol showed absence of free drug.

Example 14

Preparation of Poly(PEG-Lys)-Acyclovir Conjugate

Acyclovir succinate is prepared by heating a solution of 0.2252 g ofacyclovir (1 mmol) (Sigma), 0.200 g of succinic anhydride (2 mmoles)(Aldrich) and 0.14 mL of triethylamine in 15 mL of dry dimethylformamideat 60° C. in an oil bath for 21 hours. The solution was then cooled andthe volatile constituents were evaporated in vacuo, and the residue wastaken up in 8 mL of ice water and acidified to pH 2 with 2N HCl. A whiteprecipitate was formed that was collected by filtration, thoroughlywashed with ice water and dried in vacuo over P₂ O₅ at 40° C. to yield0.180 g (54 percent) of the product. The ester was then recrystallizedfrom methanol and characterized by IR and ¹ H NMR spectroscopy.

In a 10 mL round-bottomed flask, 0.287 g (0.133 mmol) of thepoly(PEG-Lys) having acyl hydrazine pendant functional groups of Example5 and 0.036 g (0.111 mmol) of the acyclovir succinate were dissolved in5 mL of anhydrous pyridine. The reaction mixture was cooled in an icewater bath and then 0.025 g (0.121 mmol) of DCC was added. After initialcooling for about one hour, the reaction was allowed to stir for almostthree days at room temperature. The dicyclohexyl urea that precipitatedwas removed by filtration and the product was transferred to aseparatory funnel to which 10 mL of water was added and the product wasextracted with methylene chloride. The methylene chloride extract waswashed with saturated sodium chloride solution, dried, concentrated andprecipitated with ether. About 0.140 g of product was recovered that waspurified by extraction with isopropanol. TLC in a 4:1 ratio solution ofethanol to acetic acid showed the absence of free acyclovir succinate.

Example 15

Preparation of N-Benzylcarbamate Derivative of a Copolymer of PEG andGlutamic Acid

2 g of PEG 2000 was azeotropically dried following the procedure ofExample 1 by dissolving the polymer in 30 mL of toluene in a pre-weighed50 mL round-bottomed flask provided with a stirrer. The polymer solutionwas azeotropically dried for two hours under reflux in an oil bath, thetemperature of which was maintained at 140° C. All the solvent wasdistilled off and the product was dried under vacuo. The dried PEG wasreweighed, dissolved in 5 mL of methylene chloride and stirred underargon. An equimolar amount of glutamic acid, the N-terminal of which wasprotected by a benzylcarbamate functional group (Sigma) was added. Fourtimes this amount of diisopropylcarbodiimide (Aldrich) and four timesthis amount of dimethylaminopyridinium toluene sulfonate (Aldrich) wereadded. The reaction mixture was heated- slightly to dissolve theglutamic acid. The reaction was allowed to run for 24 hours at roomtemperature with stirring. A urea precipitate formed that was removed byfiltration, and the product was precipitated by cold ether, filtered anddried under vacuum. About 1.6 g of polymer was recovered, which waspurified by reprecipitation from isopropanol. TLC in a 5:5:1 ratiosolution of toluene to acetic acid to water showed the absence of freeglutamic acid.

Example 16

Preparation of Poly(PEG-Lys) Membranes Cross-Linked by HexamethyleneDiisocyanate

A mold was prepared by clamping two square glass plates together, one ofwhich had a 5 cm diameter circular cavity. The contacting surfaces ofthe glass plates were coated with trimethylchlorosilane (Aldrich) toprevent adhesion. The mold was placed on a level surface inside a glovebox and further leveled using a carpenter's level. In a 100 mL beaker,1.5 g of the poly(PEG-Lys) having pendant acyl hydrazine groups (0.67mmol of hydrazine groups) of Example 5 was dissolved in 40 mL ofmethylene chloride. To this solution was added 1.5 g finely powderedsodium bicarbonate. The suspension was stirred for one hour and thesupernatant was tested for the presence of chloride ions with silvernitrate. A few drops of the methylene chloride solution were placed intoa test tube, the methylene chloride was evaporated, and the residue wasreacted with a few drops of silver nitrate solution acetified withnitric acid. The absence of any white turbidity indicated the completeneutralization and removal of hydrochloric acid.

The solution was then filtered and the residue was washed with methylenechloride. To the combined filtrate, 54 microliters of hexamethylenediisocyanate (56 mg, 0.67 meq of isocyanate groups) (Aldrich) was addedwith stirring. After two to three minutes of stirring, the solution waspoured into the circular cavity of the solvent casting mold. The cavityof the mold was covered with filter paper so that the solventevaporation was slow and uniform. The film was allowed to dry in theglove box for 48 hours and then peeled from the mold. The thickness ofthe membrane was measured with an electronic vernier caliper inside theglove box and was found to be about 0.1 mm.

The membranes obtained were semi-transparent and were somewhathygroscopic, curling up when exposed to moisture in ambient air. Whenplaced in water, the size of the films doubled in all dimensions,indicating a very large, swelling ratio. The swollen membranes weretransparent.

The membrane was assayed with trinitrophenyl sulfonic acid (TNBS)(Fluka) to determine the extent of cross-linking. An excess of TNBS wasused, and after reacting with the polymer, the unreacted TNBS wasallowed to react with an excess of adipic hydrazide. The IR absorbanceobtained at 500 nm was then used to calculate the amount of freehydrazides present on the cross-linked membrane. Using this method, itwas found that 80-85 percent of all available hydrazides precipitated incross-linking, leaving only 15-20 percent of unreacted hydrazides on thecross-linked membrane.

Differential Scanning Calorimietry of the cross-linked membrane showed asharp endothermic transition at 33.4° C. This is very similar to theT_(m) of the corresponding non-cross-linked poly(PEG-Lys) having pendantacyl hydrazine functional groups (34.1° C.). When the membrane washeated in an oven above the phase transition temperature, it became veryflexible but did not disintegrate. These results indicate that theproperties of PEG dominate even after copolymerization with lysine andcross-linking.

Swelling measurements of the membrane were made by two methods. Thedimensions of the dry membrane was measured and the membrane was allowedto swell in water. The increase in dimension was taken as a measure ofswelling. Alternatively, the membrane was weighed before and afterswelling and the increase in weight was taken as a measure of swelling.Both methods indicated that the membrane absorbs about 5 to 8 times itsweight of water.

Preliminary diffusion measurements were made using a small dialysiscell. p-nitroaniline was used as the diffusing solute. The membrane wasused as a partition between an aqueous solution of p-nitroaniline anddistilled water placed in the two compartments of the dialysis cell. Theabsorbance in the two compartments was measured as a function of time.These preliminary measurements showed that the rate of diffusion acrosscross-linked Poly(PEG-Lys) membranes was comparable to that of aregenerated cellulose of similar thickness.

The tensile strength of the membrane was measured using strips ofmembrane 0.07 mm thick, 5 mm wide and 50 mm long. Measurements were madeemploying both dry and swollen membranes, the results of which are shownin Table II.

                  TABLE II                                                        ______________________________________                                                Tensile  Strength   Young's                                                                              Elongation                                         at Yield at Break   Modulus                                                                              at Break                                   Membrane                                                                              (MPa)    (MPa)      (MPa)  (%)                                        ______________________________________                                        Swollen N/A      0.46       0.62    73                                        Dry     8        64         19     350                                        ______________________________________                                    

In the swollen state, the membrane behaved like a perfect elastomer. Themembrane did not exhibit a yield point and a plot of stress againststrain gave a straight line in accordance with Hooke's Law. This elasticbehavior should make them ideal materials for wound dressing and useapplications.

The stability of the membrane was investigated in acidic, basic andneutral media, the results of which are listed in Table III below. Smallspecimens of the membrane were placed in contact with a number ofaqueous solutions of varying pH at room temperature and the timerequired for the complete disappearance of the membrane was noted. Themembrane was generally found to be more stable in weakly acidic mediaand extremely unstable in alkaline media.

                  TABLE III                                                       ______________________________________                                                      TIME REQUIRED FOR                                               SOLUTION      DISAPPEARANCE                                                   ______________________________________                                        1 N HCL       5 to 8 days                                                     0.1 N HCL     No change in 8 days                                             0.01 N HCL    No change in 8 days                                             Deionized water                                                                             No change in 8 days                                             Borate (pH = 9)                                                                             5 to 8 days                                                     0.01 N NaOH   Less than 5 hours                                               0.1 N NaOH    Less than 5 hours                                               1 N NaOH      Less than 1 hour                                                ______________________________________                                    

To test the stability under physiological conditions, an acceleratedstability study was performed in which samples of membrane were exposedto phosphate buffer of pH 7.4 at 60° C. As depicted in FIG. 1, underthese conditions, the membrane lost weight at the rate of about 1percent per hour. After 60 hours, the membrane disintegrated and becamesoluble in the buffer.

Example 17

Preparation of Electrically Conductive Materials

A 10 percent solution in freshly distilled tetrahydrofuran was preparedof a mixture of lithium triflate (Aldrich) and the poly(PEG-Lys--OEt),prepared according to the procedure described in Example 2, in apolymer-electrolyte ratio of 4:1 by weight. The polymer had aweight-average molecular weight of 140,000 daltons. A film was cast fromthe solution as described above with respect to Example 16. A stickyfilm was obtained that was scraped from the glass plates, dried underhigh vacuum, and pressed into pellets at a pressure of 0.15 ton and atemperature of 27° C. This resulted in the formation of clear pellets.

Conductivity was measured using a 70 mg pellet having a thickness of 0.5mm and a 300 mg pellet having a thickness of 2.0 mm. The conductivity ofthe pellets was evaluated using standard, established techniques. First,conductivity was measured under high vacuum without exposing the pelletsto ambient conditions. The conductivity of both pellets was found to beessentially identical, and in the order of 10⁻³ ohm⁻¹ cm⁻¹. This is arelatively high value that is close to the threshold needed forcommercial applications. However, when the same samples were examined inambient air, the conductivity increased to 10⁻¹ ohm⁻¹ cm⁻¹, asignificant increase.

The temperature dependance of the ionic conductivity of the polymer wasthen measured between room temperature and 40° C., which is near themelting point of the polymer. An Arrhenius plot of conductivity vs.temperature (°K.) is shown in FIG. 2. Conductivity increases withincreasing temperature until the polymer becomes molten, at which pointconductivity remains constant as temperature increases.

Example 18

Preparation of Poly(PEG-Lys) Membranes Cross-Linked withTris(Aminoethyl) Amine

In a 100 mL beaker, 1.87 g of the PEG-bis(Succinimidyl Carbonate) ofExample 1 was dissolved in 20 ml of methylene chloride. In anotherbeaker, 82 microliters (89 mg) of tris(aminoethylamine) was dissolved in20 ml of methylene chloride. The triamine solution was added to the PEGsolution with vigorous stirring. After about five minutes, films werecast of the solution following the procedure described above withrespect to Example

Swelling measurements of the membrane were made by the two methodsdescribed above with respect to Example 16. Both methods indicated thatthe membrane absorbed about six times its weight of water.

The stability of the membrane was investigated in acidic, basic andneutral media, as described above with respect to Example 16. In sodiumhydroxide (0.01 and 0.1N) the membrane disintegrated within a few hours.In acidic media and in phosphate buffer (pH 7.4) the membrane appearedto be stable for longer periods of time. The accellerated degredationstudy of Example 16 was also performed, in which the membrane remainedintact for more than a week. An analysis of the buffer in which theaccellerated stability study was conducted revealed that during thefirst 24 hours a small amount of PEG chains had leached from thecross-linked membrane, but throughout the following 72 hours, no morePEG was leached.

Example 19

Preparation of Poly(Caprolactone) Semi-IPN's of Poly(PEG-Lys) MembranesCross-Linked by Diisocyanetohexane

The poly(PEG-Lys) membrane cross-linked by diisocyanetohexane wasprepared as in Example 16, using 210 mg of the poly(PEG-Lys) of Example5 having acyl hydrazine functional groups, dissolved in 10 mL ofmethylene chloride. The free base was formed with sodium bicarbonate,and the solution was then filtered. Prior to the addition of fourmicroliters (3.9 mg) of the hexamethylene disocyanate, 0.47 g ofpoly(caprolactone) (Union Carbide); (mw 72,000) was added to thefiltrate, which was stirred for 30 minutes to dissolve the polymercompletely. The poly(PEG-Lys) was cross-linked and films were castfollowing the procedure described above with respect to Example 16. Theresulting membrane was hydrophilic and absorbed water with anequilibrium water content of 36%, whereas film made ofpoly(caprolactone) alone is hydrophobic.

As will be readily appreciated, numerous variations and combinations ofthe features set forth above can be utilized without departing from thepresent invention as set forth in the claims. Such variations are notregarded as a departure of the spirit and scope of the invention, andall such modifications are intended to be included within the scope ofthe following claims.

What is claimed is:
 1. A polymer comprised of one or more recurringstructural units independently represented by the formula: ##STR30##wherein R₁ is a poly(alkylene oxide);R₃ and R₄ are independentlyselected from the group consisting of saturated and unsaturated,straight-chained and branched alkyl groups containing up to 6 carbonatoms and alkylphenyl groups, the alkyl portions of which are covalentlybonded to an oxygen or carbonyl group and contain up to 6 carbon atoms;and a and b are independently 0 or 1; R₅ is selected from the groupconsisting of: ##STR31## wherein --AA-- is an amino acid or peptidesequence, with the proviso that --AA-- has a free C-terminus or hydroxylgroup; D is a pendant functional group --NHZ,wherein Z is selected fromthe group consisting of hydrogen, ##STR32## an N-terminus protectinggroup and wherein a derivative of a pharmaceutically active compoundhaving an activated carboxylic acid group is directly covalently bondedto said pendant functional group of said recurring structural unit bymeans of: an amide bond formed as a result of the reaction of saidactivated carboxylic acid of said pharmaceutically active compound withsaid pendant functional group; and R₆ is selected from the groupconsisting of alkyl groups containing from two to six carbon atoms,alpha-, beta-, gamma- and omega amino acids, and peptide sequences. 2.The polymer of claim 1, wherein said pharmaceutically active compound isselected from the group consisting of chlorin e₆, cephradine,cephalothin, melphalan, penicillin V, aspirin, nicotinic acid,chemodeoxycholic acid and chlorambucil.